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Acaricidal properties of vetiver essential oil from Chrysopogon zizanioides (Poaceae) against the tick species Amblyomma cajennense and Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) Roseane Nunes de Santana Campos a , Cecília Beatriz Nascimento Lima a , Alexandre Passos Oliveira a , Ana Paula Albano Araújo b , Arie Fitzgerald Blank a , Péricles Barreto Alves c , Rafaely Nascimento Lima c , Vinícius Albano Araújo d , Alisson Silva Santana a , Leandro Bacci a,∗ a
Departamento de Engenharia Agronômica, Universidade Federal de Sergipe, Av. Marechal Rondon s/n, 49100-000, São Cristóvão, SE, Brazil Departamento de Ecologia, Universidade Federal de Sergipe, Av. Marechal Rondon s/n, 49100-000, São Cristóvão, SE, Brazil Departamento de Química, Universidade Federal de Sergipe, Av. Marechal Rondon s/n, 49100-000, São Cristóvão, SE, Brazil d Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Vic¸osa, Campus Rio Paranaíba, Rodovia BR 354, Km 310, 38810-000, Rio Paranaiba, MG, Brazil b c
a r t i c l e
i n f o
Article history: Received 9 April 2015 Received in revised form 19 August 2015 Accepted 20 August 2015 Keywords: Vetiver Ticks Toxicity Alternative control
a b s t r a c t Ticks are arthropods widely distributed in tropical and subtropical regions, which can transmit infectious agents also responsible for zoonoses. Excessive use of conventional acaricides has resulted in the onset of drug resistance by these parasites, thus the need to use alternative methods for their control. This study evaluated the acaricidal activities of Chrysopogon zizanioides (vetiver) essential oils containing different zizanoic and khuzimol (high and low acidity) acid concentrations on Amblyomma cajennense and Rhipicephalus microplus (Acari: Ixodidae). To this aims, toxicity tests of different concentrations of examined essential oils were conducted on adult females and larval stages. Results showed that the essential oils of C. zizanioides with high and low acidity reduced oviposition of females, eggs hatch and larval survival, being more effective than some commercial products widely used to control these ectoparasites. These results indicate that the C. zizanoides essential oils are promising candidates as acaricidal agents and represent also an add value to vetiver oil with high acidity, which is commercially undervalued in the cosmetic industry. © 2015 Published by Elsevier B.V.
1. Introduction Especially in tropical and subtropical regions, major economic losses are recorded annually due to tick infections and their blood-sucking habit, which confer to these arthropods medical and veterinary importance (Grisi et al., 2002). Worldwide, the economic losses caused by ticks are on the order of tens of billions dollars per year (Parizi et al., 2011). In Brazil, Rhipicephalus microplus and Amblyomma cajennense are vectors of etiological agents that cause severe diseases, like tick fever in cattle and spotted fever in humans (Olivo et al., 2008).
∗ Corresponding author at: Universidade Federal de Sergipe, Av. Marechal Rondon s/n, 49100-000, São Cristóvão, SE, Brazil. E-mail address:
[email protected] (L. Bacci).
The main method of tick control is the use of synthetic pesticides, including synthetic pyrethroids, organophosphates and amitraz (Furlong et al., 2004; Furlong and Martins, 2005; Gazim et al., 2011). However, the indiscriminated and intensive use of acaricides has caused the onset of drug-resistance phenomena by these ectoparasites (Labruna et al., 2004), environmental pollution and harm to human and animal health (Chagas et al., 2002; Chagas, 2004; Nerio et al., 2010). Thus, the search for alternative methods is of great relevance for these economic, health and ecological issues (Bacci et al., 2007). Biologically active botanical compounds represent a viable strategy for tick control, for having generally lower cost and lower toxicity to animals, humans and non-target organisms (Moreira et al., 2007; Bagavan et al., 2009). The use of plant extracts with potential acaricidal activity has been the focus of a larger number of studies, which have demonstrated repellency, oviposition inhi-
http://dx.doi.org/10.1016/j.vetpar.2015.08.022 0304-4017/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Santana Campos, R.N.d., et al., Acaricidal properties of vetiver essential oil from Chrysopogon zizanioides (Poaceae) against the tick species Amblyomma cajennense and Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.022
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bition, feed activity reduction and alteration of larval development against these arthropods (Roel, 2001). Chrysopogon zizanioides (Poaceae) (vetiver) essential oil showed bactericidal, fungicidal, insecticidal and anti-inflammatory properties (Mao et al., 2006; Bizzo et al., 2009; Danh et al., 2010). This essential oil is also widely used in the cosmetic industry as a natural fixative of volatile essences (Monteiro et al., 2011; Santos et al., 2002). There are two variations of the C. zizanioides essential oil available in the market: one with high acid value (HAV) (low quality) and the other one with low acid value (LAV) (high quality). For the cosmetic industry, its quality is related to the concentration of fatty acids and sesquiterpene zizanoic acid and khusimol (alcoholic portion) (Dantas et al., 2007; ISO 1242, 1999). The acid value of the essential oil is directly proportional to the fatty acids and zizanoic acid concentration and the reverse occurs with khusimol (Martinez et al., 2004). So far there are no data in literature demonstrating the acaricidal activity of this plant and, despite C. zizanioides HAV essential oil has lower acceptability in the perfumery industry, it is not known if it has the desired properties for the control of arthropods. Therefore, this study aimed to evaluate the acaricidal activity of C. zizanioides essential oils on A. cajennense and R. microplus. Tick reproductive parameters and larval mortality were compared with the activity of commercial products largely used in the control of these arthropod species. 2. Material and methods 2.1. Essential oils Essential oils of C. zizanioides HAV and LAV were obtained by hydrodistillation of C. zizanioides roots and acquired from the company Raros Naturals, located in Macaíba, Rio Grande do Norte, Brazil. The acid value of the C. zizanioides essential oil was measured by the amount (mg) of potassium hydroxide required to neutralize the free fatty acids contained in 1 g of essential oil (ISO 1242, 1999). 45 mg/g potassium hydroxide were used for the essential oils of C. zizanioides with high acid value (HAV) and 7.5 mg/g for oil with low acid value (LAV). 2.2. Chemical analysis of essential oils Samples of the C. zizanioides essential oils (HAV and LAV) were analyzed by chromatography using a flame ionization detector (CG–FID) and mass spectrometry (CG–EM). The chromatographic conditions were: fused silica capillary column (30 m × 0.25 mm) with a stationary phase Zebron ZB–5MS (0.25 m in film thickness); helium as a gas carrier at a flow rate of 1.8 ml/min keeping the temperature programmed in 50 ◦ C for 2 min, followed by an increase of 4 ◦ C/min until 200 ◦ C, then at 15 ◦ C up to 250 ◦ C, keeping this temperature constant for 15 min; detector temperature (or interface) 250 ◦ C; injection volume of 0.5 l in dichloromethane; partition rate of injected volume 1:100 and column pressure of 166 kPa. Conditions of the mass spectrometer: ion capture detector operating in electronic impact and energy impact of 70 eV; sweep speed 1.000; sweep range of 0.50 fragments and fragments detected on 40–500 Da. Each component of the essential oils was identified by comparing its mass spectrum, spectra and retention indexes with data reported in database (NIST 21 and NIST 107) and in the literature (Adams et al., 2008). The relative retention ratios (RRR) were determined using a calibration curve of a series of n-alkanes (C8 –C18 ) injected in the same samples chromatographic conditions. The concentration of the constituents was calculated from the integral area of the respective peaks related to the total area of all the sample constituents.
2.3. Ticks A total of 1000 engorged females of A. cajennense and R. microplus species were collected with forceps directly on horses and cattle, respectively. Animal hosts were kept isolated without application of acaricidal treatments for a minimum of 45 days. The species A. cajennense was collected in the municipality of Umbaúba (11◦ 22 27 S, 37◦ 39 27 W) and R. microplus in São Miguel do Aleixo (10◦ 23 26 S, 37◦ 22 42 W), both localities in the state of Sergipe, Brazil. The identification of the ticks was made by using specific taxonomic keys (Fletchmann, 1990). A total of 580 engorged females were used in immersion bioassays, while the remaining females were kept in biological oxygen demand (BOD) for the production of larvae, which were used in other bioassays.
2.4. Bioassay with adults A total of 580 engorged female ticks of both species (290 females for each species) kept in the laboratory were washed with distilled water and dried on paper towel for exposure to treatment. The bioassay used was adapted from Drummond et al. (1973) in which the engorged A. cajennense (198.2 ± 9.2 mg/female) and R. microplus (200.2 ± 4.0 mg/female) females were immersed for 2 min in 3 ml final volume of the solutions tested. For each bioassay, five engorged females were placed in Petri dishes (4 cm diameter, 2 cm height) lined with filter paper. The experimental design was completely randomized. All products applied were emulsified in distilled water and Triton X-100 at 2%, as surfactant agent. For the negative control females were immersed only in distilled water and Triton X-100 (2%). Ten repetitions (5 females/plate) were performed for each tick species, totalizing 100 females. Positive controls were performed using commercial products Natuneem® (Azadirachta indica oil) at the concentration of 10 l ml−1 and Butox® P CE25 (deltamethrin) at recommended doses 2 and 1 l ml−1 for A. cajennense and R. microplus, respectively. In these cases four repetitions (5 females/plate) were performed considering the two acaricides by each tick species, totalizing 80 females. For treatments containing the essential oils of C. zizanioides HAV and LAV concentrations of 1, 10, 20, 50 and 100 l ml−1 were used in four replicates (5 females/plate) for both essential oils at five concentrations by each tick species, totalizing 400 females. The plates containing treated females (=580) were covered with transparent plastic (PVC film) and kept in BOD incubator at 27 ± 1 ◦ C temperature and 75 ± 5% humidity. 14 days after oviposition the eggs were weighed to determine the yield of eggs per female (PO) (g). For each treatment, a sample (∼30 mg) was withdrawn for the evaluation of the egg hatch percentage. The eggs were placed in test tubes previously identified, sealed with PVC film and kept for 60 days under the same conditions of temperature and humidity above mentioned. After this period, the number of larvae counted in each egg mass was recorded. For the two species of ticks relations corresponding the number of eggs to its mass were laid. For this, 10 groups of about 70 mg of eggs from untreated ticks had their masses and their amount of eggs determined. Thus, it was possible to estimate the number of eggs (or estimated number of larvae) in a mass of eggs for each tick species. With these data it was possible to determine the following reproductive parameters based on the formulae according to Stendel (1980):
Please cite this article in press as: Santana Campos, R.N.d., et al., Acaricidal properties of vetiver essential oil from Chrysopogon zizanioides (Poaceae) against the tick species Amblyomma cajennense and Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.022
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i) Reproductive index (RI) = egg mass(g)/engorged females mass(g); ii) % Inhibition of oviposition (IO) = [(RI control group–RI treated group)/RI control group] × 100; iii) % of Egg hatch (EH) = (number of larvae counted/estimated number of larvae) × 100; iv) % Reproductive efficiency (RE) = RI × EC; v) % Inhibition of reproduction (IR) = [(RE control group–RE treated group)/RE control group] × 100.
2.5. Bioassay with larvae Larvae used in these assays were obtained from females kept in the laboratory. The bioassays were conducted using methodology adapted from FAO (2004). Envelopes of filter paper (2 × 2 cm) were treated with 0.1 ml of C. zizanioides HAV and LAV essential oils solutions diluted in acetone. Preliminary tests showed that this solvent has no toxicity to the larvae. In the negative control only acetone was used. Concentrations of 12 and 15 (l ml −1 ) were used for the C. zizanioides essential oils HAV and LAV, respectively. After the solvent evaporation, about 200 larvae were placed inside the envelope which was sealed with clips of “bulldog” type. The concentrations that resulted in greater than zero and less than 100% mortality were used to determine the concentration-mortality curves. The experimental design was completely randomized with three replications (an envelope ∼200 larvae) for each concentration. The envelopes were placed in Petri dishes (9 cm diameter, 2 cm height) covered with transparent plastic (PVC film) and maintained in BOD incubator at 27 ± 1 ◦ C and 75 ± 5% humidity. 48 h after setting-up bioassay, the numbers of living and dead individuals were counted with the aid of a 40x binocular stereomicroscope from the Edutec brand (40× magnification). Were considered dead the larvae that did not respond to stimulus with a brush.
2.6. Statistical analysis Analyses were performed with the aid of the statistical program SAS Institute (2001). Reproductive parameters were subjected to analysis of variance at P < 0.05 (PROC MIXED, SAS). The analyzes tested whether these parameters (variables y) differ from the essential oils (C. zizanioides HAV and LAV), concentrations of essential oils (1, 10, 20, 50 and 100 l ml−1 ), tick species (A. cajennense and R. microplus) and essential oils interactions x concentrations, essential oils x species and concentrations x species (variables x). The effect of C. zizanioides HAV and LAV essential oils and of the positive controls in reproductive parameters PO, IP EO and ER for both tick species were compared with the negative control (H2 O + Triton 2%) by Dunnett’s test at P < 0.05 (PROC GLM, SAS). And the differences among the average of reproductive parameters IO and CR were compared by Tukey test at P < 0.05 (PROC GLM, SAS). The results of larval mortality were corrected in what relates to the mortality occurred in the evidence using Abbott’s formula (1925). Probit analyses were performed to determine the concentration-mortality curves of the C. zizanioides HAV and LAV essential oils for A. cajennense and R. microplus larvae. Only the curves that showed Probit’s distribution through the test x2 with probability greater than 0.05 were accepted (PROC PROBIT, SAS). From these curves were estimated lethal concentrations to cause mortality of 1, 50 and 99% of the population (LC01; LC50 e LC99 ) and their respectively confidence intervals at 95% probability. For all the analyses of variance and probit, the assumptions of normality and homogeneity of variance were checked (PROC UNIVARIATE, PROC GPLOT, SAS), not requiring data transformation.
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3. Results 3.1. Essential oils characterization The composition of the C. zizanioides essential oils analyzed on GC–MS and GC–FID is shown in Table 1. All compounds identified in the C. zizanioides HAV and LAV oils are sesquiterpenes, being 19.5 and 7.8% hydrocarbons and 55.9 and 7.6% oxygenated, respectively. The khusimol the major compound in the C. zizanioides HAV and LAV oils (16.28 and 19.39%, respectively). The other major constituents in the C. zizanioides HAV and LAV essential oils were isovalencenol (8.94 and 13.20%); ␣-vetivone (7.43 and 5.38%); ␣-cadinol (6.12 and 8.54%) and -vetivone (5.30 and 4.61%). The zizanoic acid, one of the compounds related to this essential oil quality, has showed concentrations of 1.30 and 0.46% in oils HAV and LAV, respectively. 3.2. Bioassays with adults The eggs production per female (g), the reproductive index and the% inhibition of oviposition of engorged females in the different treatments are shown in Table 2. The results of the variance analysis showed that there were significant differences in the concentrations of the applied oils, considering the eggs production per female (F4;60 = 11.59; P < 0.001), the reproductive index (F4;60 = 18.14; P < 0.001) and the % inhibition of oviposition (F4;60 = 19.16; P < 0.001). However, these same parameters showed no significant differences between the essential oils (egg production/female: F1;60 = 2.23; P = 0.140; reproductive index: F1;60 = 1.24; P = 0.270, and% inhibition of oviposition: F1;60 = 0.87; P = 0.354) and there were no differences between the species of ticks (egg production/female: F1;60 = 2.46; P = 0.122; reproductive index: F1;60 = 2.34; P = 0.131, and% inhibition of oviposition: F1;60 = 0.02; P = 0.878) and their interactions. There was a significant reduction in the egg production per female and the reproductive index at concentrations of 20, 50 and 100 l ml−1 of the C. zizanioides essential oils for both tick species. At the lowest concentration (1 l ml−1 ), both essential oils were able to reduce R. microplus eggs production, while C. zizanioides LAV essential oil reduced A. cajennense % reproductive index (Table 2). Natuneem® and Butox® P CE25 commercial products did not affect egg production per female. With the exception of A. cajannense reproductive index treated with Butoxg P CE25, the other results of these products did not differ from the control (Table 2). The decrease in egg production resulted in % inhibitions of oviposition ranged from 20 to 82% for A. cajannense and from 17 to 95% for R. microplus. The greatest % inhibitions of oviposition were observed at concentrations of 50 and 100 l ml−1 (Table 2). Percentage of egg hatch, the % reproductive efficiency and the % inhibition of reproduction of engorged females of A. cajennense and R. microplus after exposure to treatments are described in Table 3. The result of the variance analysis showed that there were differences in the concentrations of essential oils in relation to % of egg hatch (F4;60 = 16.25; P < 0.001), % reproductive efficiency (F4;60 = 75.35; P < 0.001) and% inhibition of reproduction (F4;60 = 75.87; P < 0.001). However, these parameters did not differ between the essential oils (% of egg hatch: F1;60 = 0.07; P = 0.798; % reproductive efficiency: F1;60 = 3.15; P = 0.081, and % inhibition of reproduction: F1;60 = 2.96; P = 0.091), nor there were significant differences between the tick species (% egg hatch: F1;60 = 0.74; P = 0.393; % reproductive efficiency: F1;60 = 2.08; P = 0.154; % inhibition of reproduction: F1;60 = 0.06; P = 0.811) and their interactions. Egg hatch was significantly reduced from 20 to 50 l ml−1 (A. cajennense) and 50 and 10 l ml−1 (R. microplus) when females were treated with C. zizanioides HAV and LAV essential oils, respectively. Natuneem® and Butox® P CE25 commercial products did not sig-
Please cite this article in press as: Santana Campos, R.N.d., et al., Acaricidal properties of vetiver essential oil from Chrysopogon zizanioides (Poaceae) against the tick species Amblyomma cajennense and Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.022
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Table 1 Chemical composition of the Chrysopogon zizanioides essential oils with high (HAV) or low (LAV) value of acidity characterized by GC/MS and GC/FID. Essential oil and average ± standard error of the peak area (%)b
RIa
Compound
Prezizaene Khusimene ␣-Amorphene ␣-Vetispirene -Vetispirene ␥-Amorphene ␦-Amorphene ␦-Cadinene ␥-Vetivenene -Vetivenene Khusimone Junenol Eremoligenol ␣-Cadinol Epi-zizanone Germacra-4(15), 5,10(14)-trien-1-␣-ol Vetiselinenol Khusimol ␣-Costol Isovalencenol Zizanoic acid -Vetivone Eudesm-7(11)-en-4-ol ␣-Vetivone Total detected (%)
1454 1459 1486 1492 1497 1500 1513 1528 1536 1561 1611 1628 1641 1662 1678 1686 1733 1755 1777 1801 1819 1831 1842 1856
HAV(45.0 mg/g)c
LAV(7.5 mg/g)c
1.16 ± 0.06 1.45 ± 0.00 4.28 ± 0.13 2.22 ± 0.10 2.68 ± 0.02 1.15 ± 0.08 1.15 ± 0.10 1.52 ± 0.05 1.18 ± 0.05 2.75 ± 0.15 – 4.26 ± 0.09 1.31 ± 0.10 6.12 ± 1.29 2.63 ± 0.12 – 2.80 ± 0.11 16.28 ± 0.34 – 8.94 ± 0.31 1.30 ± 0.17 5.30 ± 0.15 1.52 ± 0.03 7.43 ± 0.25 77.38
0.60 ± 0.03 0.72 ± 0.02 1.96 ± 0.03 0.94 ± 0.04 1.25 ± 0.02 0.68 ± 0.03 – 0.81 ± 0.02 – 0.84 ± 0.19 1.48 ± 0.02 3.92 ± 0.08 1.28 ± 0.04 8.54 ± 0.04 1.84 ± 0.06 0.84 ± 0.04 5.05 ± 0.03 19.39 ± 0.15 0.42 ± 0.01 13.20 ± 0.01 0.46 ± 0.03 4.61 ± 0.08 1.19 ± 0.05 5.38 ± 0.03 75.39
Dashes indicate that the compound was not found. a Retention index calculated using the equation Van den Dool and Kratz 1963 in relation to a homologous n-alkanes series (nC9 –nC18 ). b Compound content values obtained by the average of three different determinations obtained by GC/MS and GC/FID. c Values indicate the amount of potassium hydroxide required to neutralize the free fatty acids present in the C. zizanioides essential oils. Table 2 Average (±SE) egg production per female (g), reproductive index and % inhibition of oviposition (%) of Amblyomma cajennense and Rhipicephalus microplus engorged females treated with the Chrysopogon zizanioides essential oil with high (HAV) and low (LAV) acid value. Treatment
(l ml−1 )
Eggs production per female (g)a HAV
LAV
Control (H2 O + Triton 2%) C. zizanioides (1) C. zizanioides (10) C. zizanioides (20) C. zizanioides (50) C. zizanioides (100) Natuneem® (10)c Butox® P CE25 (2)c
0.146 ± 0.006 0.135 ± 0.034 0.121 ± 0.028 0.055 ± 0.002 * 0.033 ± 0.013 * 0.033 ± 0.015 * 0.129 ± 0.010 0.086 ± 0.015
0.146 ± 0.006 0.088 ± 0.028 0.061 ± 0.035 * 0.038 ± 0.004 * 0.051 ± 0.033 * 0.016 ± 0.010 * 0.129 ± 0.010 0.086 ± 0.015
Control (H2 O + Triton 2%) C. zizanioides (1) C. zizanioides (10) C. zizanioides (20) C. zizanioides (50) C. zizanioides (100) Natuneem® (10)c Butox® P CE25 (1)c
0.121 ± 0.008 0.080 ± 0.010 * 0.065 ± 0.014 * 0.063 ± 0.014 * 0.036 ± 0.007 * 0.009 ± 0.003 * 0.093 ± 0.011 0.110 ± 0.004
0.121 ± 0.008 0.079 ± 0.010 * 0.070 ± 0.012 * 0.053 ± 0.010 * 0.045 ± 0.019 * 0.005 ± 0.003 * 0.093 ± 0.011 0.110 ± 0.004
a b c
Reproductive indexa HAV A. cajennense 0.634 ± 0.024 0.495 ± 0.042 0.506 ± 0.041 0.371 ± 0.045 * 0.232 ± 0.094 * 0.173 ± 0.061 * 0.528 ± 0.024 0.359 ± 0.031 * R. microplus 0.535 ± 0.021 0.386 ± 0.060 0.360 ± 0.084 * 0.304 ± 0.057 * 0.191 ± 0.030 * 0.049 ± 0.017 * 0.459 ± 0.033 0.495 ± 0.024
% Inhibition of ovipositionb LAV
HAV
LAV
0.634 ± 0.024 0.393 ± 0.047 * 0.306 ± 0.111 * 0.296 ± 0.016 * 0.240 ± 0.101 * 0.114 ± 0.075 * 0.528 ± 0.024 0.359 ± 0.031 *
– 21.82 ± 6.69 b 20.16 ± 6.41 b 41.39 ± 7.10 ab 63.32 ± 14.77 a 72.77 ± 9.63 a 16.62 ± 3.73 b 43.28 ± 4.82 ab
– 37.95 ± 7.45 ab 51.74 ± 17.48 ab 53.31 ± 2.60 ab 62.06 ± 15.97 ab 81.96 ± 11.88 a 16.62 ± 3.73 b 43.28 ± 4.82 ab
0.535 ± 0.021 0.444 ± 0.038 0.392 ± 0.039 0.290 ± 0.058 * 0.249 ± 0.106 * 0.026 ± 0.015 * 0.459 ± 0.033 0.495 ± 0.024
– 28.16 ± 10.95 bc 34.37 ± 14.34 bc 43.13 ± 10.60 bc 64.38 ± 5.65 ab 90.81 ± 3.09 a 14.20 ± 6.22 c 8.07 ± 4.12 c
– 17.05 ± 7.11 bc 26.77 ± 7.36 bc 45.84 ± 10.76 bc 53.46 ± 19.78 ab 95.11 ± 2.84 a 14.20 ± 6.22 bc 8.07 ± 4.12 c
Values followed by ‘*’ differ from the control (H2 O + Triton 2%), by Dunnett’s test at P < 0,05. Values followed by the same letter in the column have averages that do not differ among themselves by the Tukey’s test at P < 0,05. Commercial products used as positive controls in the recommended concentrations for each species.
nificantly reduce this parameter in both species (Table 3). When exposed to the lower concentration of C. zizanioides essential oils, reproductive efficiency was reduced for both tick species, except for C. zizanioides LAV on R. microplus females that affected this parameter from the concentration of 10 l ml−1 (Table 3). The decrease in egg production and egg hatch ranged from 26 to 95% for A. cajennense and from 17 to 100% for R. microplus (Table 3). The larger inhibition of reproduction was observed at concentrations of 50 and 100 l ml−1 (Table 3).
3.3. Bioassays with larvae Mortality curves of A. cajennense and R. microplus larvae observed for each concentration of C. zizanioides HAV and LAV essential oils are shown in Table 4. Both the essential oils showed acaricidal activity on larvae of the two tick species, with LC50 ranging from 1.175 to 4.05 l ml−1 . C. zizanioides LAV essential oil showed a higher toxicity to A. cajennense, while R. microplus was more susceptible to C. zizanioides HAV essential oil
Please cite this article in press as: Santana Campos, R.N.d., et al., Acaricidal properties of vetiver essential oil from Chrysopogon zizanioides (Poaceae) against the tick species Amblyomma cajennense and Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.022
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Table 3 Average (±SE) % of egg hatch, % reproductive efficiency and % inhibition of reproduction of engorged Amblyomma cajennense and Rhipicephalus microplus females treated with the Chrysopogon zizanioides essential oil with high (HAV) and low (LAV) acid value. Treatment
(l ml−1 )
% Egg hatcha HAV
LAV
Control (H2 O + Triton 2%) C. zizanioides (1) C. zizanioides (10) C. zizanioides (20) C. zizanioides (50) C. zizanioides (100) Natuneem® (10)c Butox® P CE25 (2)c
84.46 ± 3.83 79.74 ± 8.76 59.00 ± 7.36 32.91 ± 12.03 * 35.04 ± 16.60 * 20.66 ± 9.08 * 80.98 ± 2.25 69.08 ± 5.57
84.46 ± 3.83 74.50 ± 9.48 56.14 ± 4.15 59.59 ± 11.76 28.70 ± 17.62 * 25.50 ± 9.52 * 80.98 ± 2.25 69.08 ± 5.57
Control (H2 O + Triton 2%) C. zizanioides (1) C. zizanioides (10) C. zizanioides (20) C. zizanioides (50) C. zizanioides (100) Natuneem® (10)c Butox® P CE25 (1)c
90.23 ± 3.06 73.64 ± 7.43 67.89 ± 23.58 60.83 ± 20.94 24.95 ± 24.95 * 0.00 ± 0.00 * 90.48 ± 0.82 67.03 ± 3.60
90.23 ± 3.06 90.62 ± 2.04 47.94 ± 2.01 * 26.30 ± 9.73 * 18.68 ± 10.78 * 12.09 ± 7.07 * 90.48 ± 0.82 67.03 ± 3.60
a b c
% Reproductive efficiencya
% Inhibition of reproductionb
HAV
LAV
HAV
LAV
53.51 ± 2.06 29.29 ± 3.51 * 17.16 ± 6.22 * 17.63 ± 0.98 * 6.90 ± 2.90 * 2.92 ± 1.92 * 42.78 ± 1.92 24.82 ± 2.11 *
– 26.19 ± 6.32 cd 44.22 ± 4.48 bc 77.16 ± 2.77 a 84.78 ± 6.13 a 93.34 ± 2.36 a 20.05 ± 3.58 d 53.61 ± 3.94 b
– 45.27 ± 6.57 bc 67.93 ± 11.62 ab 67.06 ± 1.83 ab 87.11 ± 5.43 a 94.55 ± 3.59 a 20.05 ± 3.58 c 53.61 ± 3.94 b
48.26 ± 1.91 40.20 ± 3.45 18.78 ± 1.89 * 7.62 ± 1.51 * 4.65 ± 1.98 * 0.32 ± 0.18 * 41.52 ± 3.01 33.19 ± 1.64 *
– 41.12 ± 9.15 cd 49.33 ± 11.84 c 61.66 ± 7.15 bc 90.15 ± 1.56 ab 100.00 ± 0.00 a 14.01 ± 6.20 d 31.22 ± 3.40 cd
– 16.69 ± 7.14 c 61.09 ± 3.91 b 84.21 ± 3.14 a 90.37 ± 4.09 a 99.34 ± 0.38 a 14.016.20 c 31.22 ± 3.40 c
A. cajennense 53.51 ± 2.06 39.49 ± 3.38 * 29.84 ± 2.40 * 12.22 ± 1.48 * 8.14 ± 3.28 * 3.56 ± 1.26 * 42.78 ± 1.92 * 24.82 ± 2.11 * R. microplus 48.26 ± 1.91 28.41 ± 4.41 * 24.45 ± 5.71 * 18.50 ± 3.45 * 4.75 ± 0.75 * 0.00 ± 0.00 * 41.52 ± 3.01 33.19 ± 1.64 *
Values followed by “*” differ from the control (H2 O + Triton 2%), by Dunnett’s test at P < 0,05. Values followed by the same letter in the column have averages that do not differ among themselves by the Tukey’s test at P < 0,05. Commercial products used as positive controls in the recommended concentrations for each species.
Table 4 Acaricidal activities (LC01 , LC50 and LC99 ) of Chrysopogon zizanioides essential oils with high (HAV) or low (LAV) acid values on Amblyomma cajennense and Rhipicephalus microplus larvae after 48 h of exposure. C. zizanioides essential oil
Na
LC01 (l ml−1 )b (CI95)
HAV LAV
6421 9996
0.331 (0.204–0.461) 0.630 (0.527–0.713)
HAV LAV
7722 9067
0.273 (0.119–0.441) 1.702 (1.415–1.945)
a b
LC50 (l ml−1 )b (CI95) A. cajennense 2.076 (1.853–2.311) 1.175 (1.117–1.226) R. microplus 1.552 (1.252–1.786) 4.050 (3.831–4.287)
LC99 (l ml−1 )b (CI95)
Slope
x
P
13.030(9.596–20.390) 2.190 (2.002–2.490)
2.92 8.61
4.48 4.3
0.104 0.114
8.823 (6.630–14.320) 9.638 (8.386–11.680)
3.09 6.19
1.88 2.81
0.608 0.244
Number of tick larvae used in bioassays. LC: lethal concetration; CI: confidence interval at 95%.
(LC50 = 1.55 l ml−1 ). The slopes of the curves were higher for C. zizanoides LAV essential oil for both species (Table 4).
4. Discussion In previous studies, essential oils from a number of plant species showed acaricidal activity against ticks. In most of these studies the acaricidal activity of plant-derived products was evaluated on R. microplus (Mendes et al., 2011). Among them, essential oils of Copaifera reticulate, the copaíba tree, and the incense stick plant Tetradenia riparia, showed acaricidal activities also at a low concentrations, by reducing R. microplus egg number and weight, egg hatch and causing mortality of R. microplus larvae (Fernandes et al., 2007; Gazim et al., 2011). Silva et al. (2009) tested the toxicity of Piper aduncum (matico) from the Amazon forest for R. microplus larvae and demonstrated that the mortality of larvae of this species was due to an oil component, a phenylpropanoid derivative. According to Cardona et al., (2007) the use of Sapindus saponaria essential oil is a promising tool for the tick control in cattle, because it is able to reduce tick reproductive efficiency. In the present study, the C. zizanioides HAV and LAV essential oils have showed acaricidal properties against the tick species A. cajannense and R. microplus. The choice of vetiver essential oil from Chrysopogon zizanioides (Poaceae) was based on its bactericide, fungicide, insecticide and anti-inflammatory properties (Mao et al., 2006; Bizzo et al., 2009; Danh et al., 2010). Although other studies have demonstrated the antimicrobial and insecticidal potential of this essential
oil (Hammer et al., 1999; Zhu et al., 2003), its acaricidal activity was never evaluated before. In this study, chemical analysis indicated that the sesquiterpene fraction is dominant in the composition of vetiver essential oils, though khusimol is the main active compound. Previous studies have demonstrated that monoterpenes and sesquiterpenes have acaricidal activity against R. microplus (Facey et al., 2005). Terpenes are also known to have pesticidal activity against the rice weevil (Sitophilus oryzae), being able to alter development and physiology and to inhibit the reproduction of this arthropod species. Price and Berry (2006) showed that these compounds may act as neurotransmitters, neuro-modulators and neuro-hormones crucial for regulating oviposition in various arthropods (Cossío-Bayúga et al., 2012), including ticks (Rodriguez-Valentin et al., 2006; CossíoBayúga et al., 2012). The oviposition reduction of plant essential oils has been attributed to a reduction in the amount of endogenous proteins and lipids in the ovaries and oocytes of females (Williams, 1993; Williams et al., 1997). Terpenes can even act as cholinesterase inhibitors (Loizzo et al., 2010) which causes arthropods paralysis and death (Ribeiro et al., 2012). In this study C. zizanioides essential oils reduced the production of eggs by females tick and the hatch of eggs, with consequent lower rates of tick reproductive efficiency. In addition, the rate of reduction of reproductive capacity observed in ticks treated with C. zizanioides essential oils was higher than that observed with the reference commercial products Natuneem® (A. indica oil) and Butox® P CE25 (deltamethrin). In the case of A. cajannense for Butox® P CE25 and A. cajannense and R. microplus for Natuneem® , lower
Please cite this article in press as: Santana Campos, R.N.d., et al., Acaricidal properties of vetiver essential oil from Chrysopogon zizanioides (Poaceae) against the tick species Amblyomma cajennense and Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.022
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tick mortality rates were also observed when compared to those for C. zizanioides essential oils. Concerning the activity of vetiver essential oils on tick larval stages, results here obtained showed that these essential oils have significant potential as acaricides for the control A. cajannense and R. microplus larvae if compared with the larvicidal activity of other plant-derived compounds. In fact, in previous studies bitter cinnamon essential oil, Drymis brasiliensis, containing predominantly sesquiterpenes caused 100% mortality of R. microplus larvae at the concentrations of 2.5% (Ribeiro et al., 2008) while Eucalyptus citridodora and Cymbopongon nardus essential oils caused 53.1 and 61.1% mortality of A. cajennense larvae at the concentration of 50% (Clemente et al., 2010). These are similar or higher concentrations when compared to those of C. zizanioides essential oils found effective in the present study. According to Bittencourt et al., (1990), A. cajennense requires higher concentrations of acaricide than R. microplus. However, in general, similar vetiver essential oil concentrations were active against these two tick species. In this study it also demonstrates that the two different oils of vetiver (high and low acidity) show similar acaricide activities. According to the quality standards, the lower the concentration of zizanoic and higher the Khusimol acid, the better the quality of vetiver essential oil (Dantas et al., 2007; Arrigoni-Blank et al., 2011). Vetiver essential oil produced in Brazil has high zizanoic acid values when compared to the oil produced in Haiti, Bourbon and Java (Rocha, 2006). Thus, the present study shows that vetiver oil with high acidity has a potential as acaricide for ticks, adding value to this oil of little interest to the cosmetic industry. Previous studies demonstrated that ethanolic extracts of vetiver roots have low toxicity to rats (Barros et al., 2009). It has been shown that essential oils can act in octapaminergic system in insects (Rattan, 2010). Thus, the absence of octapamine receptors in vertebrates contributes to its relatively low toxicity to mammals (Isman, 2000; Rattan, 2010). Beside this the essential oils have high volatility and consequent low persistence in the environment (Rattan, 2010). Therefore, vetiver essential oils may possibly have low toxicity for the environment and vertebrates. 5. Conclusion In conclusion, C. zizanioides HAV and LAV may represent potential alternative acaricides for the control of ticks and a model for the synthesis of new acaricides. References Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–267. Adams, R.P., Robert, P., Sanko, N., Dennis, A.J., Park, S., Provin, T.L., Habte, M., 2008. Comparison of vetiver root essential oils from cleansed (bacteria- and fungus-free) non-cleansed (normal) vetiver plants. Bio. Sys. Ecol. 36, 177–182. Arrigoni-Blank, M.F., Santos, A.V., Blank, A.F., 2011. Organogênese direta e aclimatizac¸ão de plantas de patchouli. Hortic. Bras. 29, 145–150. Bacci, L., Picanc¸o, M.C., Fernandes, F.L., Silva, N.R., Martins, J.C., 2007. Estratégias e táticas de manejo dos principais grupos de ácaros e insetos-praga em hortalic¸as no Brasil. In: Zambolin, L., Lopes, C.A., Picanc¸o, M.C., Costa, H. (Eds.), Manejo Integrado de Doenc¸as e Pragas. Suprema Gráfica e Editora Ltda, Visconde do Rio Branco, Brasil, pp. 463–504. Bagavan, A., Kamara, J.C., Elango, G., Zahir, A.A., Rahuman, A.A., 2009. Adulticidal and larvicidal efficacy of some medicinal plant extracts against tick, fluke and mosquitoes. Vet. Parasitol. 166, 286–292. Barros, G.C., Tresvenzol, M.F.L., Cunha, L.C., Ferri, P.H., Paula, J.R., Bara, M.T.F., 2009. Composicao Quimica, Atividade Antibacteriana e Avaliac¸ão da Toxicidade Aguda de Vetiveria zizanoides L. Nash (Poaceae). Lat. Am. J. Pharm. 28 (4), 531–537. Bittencourt, A.J., Fonseca, A.H., Faccini, J.L.H., Bueno, B.H., 1990. Comportamento do R. microplus (Canestrini, 1887) (Acari) em infestac¸ões artificais e naturais em diferentes hospedeiros. Arq. Univ. Fed. Rural. R. J. 13 (2), 173–182. Bizzo, H.R., Hovell, A.M.C., Rezende, C.M., 2009. Óleos essenciais no Brasil: aspectos gerais, desenvolvimento e perspectivas. Quim. Nova 32, 588–594. Cardona, Z.E., Torres, R.F., Echeversi, L.F., 2007. In vitro de los extractos crudos de Sapindus saponaria sobre hembras ingurgitadas de Boophilus microplus (Acari: Ixodidae). Sci. Tech. 33, 51–54.
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Please cite this article in press as: Santana Campos, R.N.d., et al., Acaricidal properties of vetiver essential oil from Chrysopogon zizanioides (Poaceae) against the tick species Amblyomma cajennense and Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.022
