Parasitol Res (2009) 105:1093–1097 DOI 10.1007/s00436-009-1530-x
ORIGINAL PAPER
Acaricidal efficacy of thymol on engorged nymphs and females of Rhipicephalus sanguineus (Latreille, 1808) (Acari: Ixodidae) Caio Márcio de Oliveira Monteiro & Erik Daemon & Mateus Aparecido Clemente & Leonardo dos Santos Rosa & Ralph Maturano
Received: 25 May 2009 / Accepted: 8 June 2009 / Published online: 27 June 2009 # Springer-Verlag 2009
Abstract The present work was aimed at evaluating the “in vitro” efficacy of different concentrations of thymol on engorged nymphs and females of Rhipicephalus sanguineus. The nymphs were separated in seven groups and immersed in different concentrations of thymol (0.25%, 0.5%, 1.0%, 1.5%, and 2.0%) for 5 min. A control group was established (water+dimethylsulfoxide) together with a positive control group (Amitraz*), and mortality was evaluated after 15 days. In order to perform the experiment with females, the same methodology was used, and the following concentrations were tested: 1.0%, 1.5% and 2.0%. After 5 min of immersion, the engorged females were fixed on Petri dishes with the aid of a double-sided adhesive tape and kept in a climatized room regulated at 27±1°C and UR >80±10%, and different parameters referring to the biology of the nonparasitary phase were evaluated daily. The values found for thymol efficacy on nymphs were 0.0%, 100%, 100%, 100%, and 100% in concentrations of 0.25%, 0.5%, 1.0%, 1.5%, and 2.0%, respectively. In the experiment with engorged females, thymol did not induce any significant alterations (p0.05). The concentration of thymol 2% was the one that showed a better efficacy (41%). It was concluded that thymol had a more accentuated deleterious effect on engorged nymphs, and it might be a promising supporting agent for the control of this ixodid. C. M. de Oliveira Monteiro (*) : E. Daemon : M. Aparecido Clemente : L. dos Santos Rosa : R. Maturano Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora, Brazil e-mail:
[email protected]
Introduction Rhipicephalus sanguineus (Latreille 1808) (Acari: Ixodidae), more commonly known as “the brown dog tick,” is an ixodid that has a broad geographic distribution, whose medical– veterinary importance is characterized by direct (host spoliation) and indirect (transmission of pathogens) damages in dogs (Labruna 2004; Dantas-Torres 2008). This tick also assumes importance in the transmission of diseases to humans. In the Mediterranean region, it was incriminated as a vector of Rickettsia conori, etiologic agent of Boutonneuse Fever (Sousa and Bacelar 2004; Matsumoto et al. 2005), and recently, it was also incriminated in the USA as a vector of R. rickettsii, etiologic agent of Rocky Mountain spotted fever (Demma et al. 2005). In Brazil, this ixodid is considered a potential vector of this latter agent (Rozental et al. 2002), a fact that has been reinforced by recent reports on human parasitism by this tick (Dantas-Torres et al. 2006; Louly et al. 2006; Guglielmone et al. 2006). The use of synthetic acaricides is the traditional method for the control of this arthropod; yet, currently, there are reports related to R. sanguineus populations resistant to different chemical bases commercially available (Miller et al. 2001; Martins et al. 2006; Borges et al. 2007). Besides the aspect of the resistance, the demand in the search for new pest-control alternatives is increasing, aiming to minimize the use of chemicals, in order to reduce the quantity of residues in the environment (Baladrin et al. 1985; Chagas 2004). Therefore, the search for products of vegetable origin has been gaining more and more ground, as they present lower risk of environment contamination and slower resistance development rate (Chagas 2004). Thymol (=5meil-2-isopropil-1fenol) is a volatile, refringent monoterpenoid found in plants
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of the Lamiacea family. This monoterpene has already had its bactericidal, fungicidal, nematicidal, inseticidal, and molluscicidal potential evident to different authors (Imdorf et al. 1995; Paster et al. 1995; Ji et al. 2005; Mansour et al. 2000; Calvet et al. 2001; Ferreira 2005). However, the deleterious action of thymol on ticks has only been recently demonstrated in studies by Novelino et al. (2007) and Daemon et al. (2009) on larvae of Boophilus microplus (Canestrini 1887) and R. sanguineus, respectively. Thus, the objective of the present work was to evaluate the acaricidal efficacy of different concentrations of thymol on engorged nymphs and females of R. sanguineus.
Materials and methods The study was developed at the Advanced Laboratory of Zoology of the Department of Zoology of the Federal University of Juiz de Fora, Minas Gerais, Brazil. In order to perform the work, engorged nymphs and females of R. sanguineus were obtained from a colony through artificial infestations of rabbits Oryctolagus cuniculus (L., 1758), according to a methodology proposed by Neitz et al. (1971). Due to their low water solubility, thymol solutions were emulsified under heating (60°C) in aqueous dimethylsulfoxide (DMSO, 1%).
female one repetition), and then submitted to immersion for 5 min in thymol solution concentrations of 1.0%, 1.5%, and 2.0%, according to Drummond et al. (1973). A control group was established (water+DMSO) and also a positive control group (Amitraz–Triatox®, Coopers), each one containing ten females. After immersion, the engorged females were fixed, with the aid of double-sided adhesive tape, in dorsal decubitus position in Petri dishes properly identified and conditioned in a climatized room at 27±1°C and UR >80±10% for the monitoring of the following parameters: initial weight, weight of engorged females; final weight, obtained 3 days after the end of posture; alteration the weight, initial weight−final weight; egg mass weight, total egg mass weight of each female; preoviposition period, period which comprises the day between the fall of the engorged female until the beginning of the posture; hatching percentage (%EP), visual estimates of eclodid larvae in relation to the eggs mass of each female; egg production index (EPI), obtained according to a formula proposed by Bennett (1974), where EPI=egg mass×100/initial weight; nutritional index (NI), obtained according to the formula proposed by Bennett (1974), where NI=egg mass×100/(initial weight−final weight); estimate of reproduction (ER), obtained by the formula, (PP/PI)×%EC×20.000 (Drummond et al. 1973); percentage of control (%C), obtained according to the formula of Drummond et al. (1973), %C=[(ER control group−ER treated group)/ER control group]×100.
Experiment 1 Statistical analysis The nymphs obtained through artificial infestations of rabbits were separated in groups and immersed in aqueous solutions for 5 min, testing the following concentrations of thymol 0.25%, 0.5%, 1.0%, 1.5%, and 2.0%. For each treatment, ten repetitions were performed, each repetition with five nymphs (each repetition=an experimental unity). A control group was established (water+DMSO) and also a positive control group (Amitraz*), each one with ten repetitions. After the immersion period, each repetition containing five engorged nymphs was kept in test tubes properly identified, sealed with hydrophilic cotton, and kept in a climatized room regulated at 27±1°C and UR >80± 10%. After 15 days, the mortality rate was evaluated. The mortality rate for these treatments was obtained according to the following formula: Mortalityð%Þ ¼
dead nymphs 100 Total tick
Experiment 2 In order to perform the test, 80 females were used, which were weighted and separated in three groups of 20 (each
The statistical analysis was performed using the software Biostat version 5.0. The percentage values were transformed into √arcsen x. The median values of each treatment were analyzed by ANOVA and Tukey test (p0.05) (Table 2). The values
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Table 1 Mean mortality of engorged nymphs of Rhipicephalus sanguineus treated with different concentrations of thymol under laboratory conditions (27 ± 1°C and UR >80 ± 10%) (Advanced Laboratory of Zoology, UFJF) Concentrations of thymol
Mortality percentage (X±SD)
Water+DMSO
0.0a ±0.0 n=(10) 0.0a ±0.0 n=(10) 100.0b ±0.0
Thymol 0.25% Thymol 0.50%
n=(10) 100.0b ±0.0 n=(10) 100.0b ±0.0 n=(09) 100.0b ±0.0 n=(10) 100.0b ±0.0 n=(10)
Thymol 1.0% Thymol 1.5% Thymol 2.0% Amitraz
In relation to the parameters pre-oviposition period, hatching percentage, EPI, and NI, no one of the groups treated with different concentrations of thymol presented significant differences (p>0.05) in relation to the control. The females submitted to treatment with amitraz did not perform posture; thus, the analysis of these parameters was not carried out (Table 3). In relation to the engorged females, the best efficacy for the control was of 41.41%, obtained in concentration of 2.0%, while in concentrations of 1.0% and 1.5%, the efficacy was of 22.28% and 25.70%, respectively (Fig. 1).
Discussion
Means followed by the same letters in the same column do not differ statistically at a significance level of 5%. (Kruskal–Wallis/Student– Newman–Keuls) n, number of experimental units per treatment; experimental unit, five engorged nymphs.
referring to the final female weight of the treated groups were significantly different (p>0.05) from the control group, different from what was observed in relation to the parameters alteration in female weight and egg mass weight, where only the group treated with amitraz differed significantly from the control (p>0.05; Table 2).
One of the biggest problems in controlling different tick species is the resistance that they show to chemical acaricides, but vegetable origin substances have been demonstrating relative efficiency to the control of these ectoparasites (Chagas 2004). The results found for the engorged nymphs of R. sanguineus in the present work corroborate these data, once the concentration of 0.5% was enough to cause mortality of 100%, which is similar to that obtained in the positive control group. The acaricide effect of thymol on immature stages of ticks was also demonstrated by Novelino et al. (2007) and Daemon et al. (2009), where the utilization of this monoterpene resulted in high mortality rate of larvae of B. microplus and R. sanguineus, respectively. In the test with engorged females, thymol effect did not induce significant alterations for most of the analyzed parameters, and the best efficacy for treatments
Table 2 Mean weight of engorged females (mg) and egg mass weight (mg) of Rhipicephalus sanguineus treated with different concentrations of thymol, under laboratory conditions (27±1°C e UR>80±10%) (Advanced Laboratory of Zoology, UFJF) Concentrations of thymol
Initial weight (mg)
Final weight (mg)
Alteration weight (mg)
Egg mass weight (mg)
Water+DMSO
131.0ª±29.1 (9) 131.4ª±24.9 (20) 131.6ª±30.1 (20) 133.9ª±25.4 (20)
19.6ª±7.1 (9) 43.7b ±34.0 (20) 42.2b ±41.5 (20) 60.2bc ±46.3 (20)
111.3ª±25.6 (9) 87.6ª±41.7 (20) 89.2ª±39.1 (20) 73.6ab ±53.1 (20)
72.1ª±14.3 (9) 53.7ª±32.3 (20) 55.9ª±33.0 (20) 47.4ª±38.4 (20)
135.7ª±25.6 (10) ANOVA
85.5c ±24.0 (10) Kruskal–Wallis/Student– Newman–Keuls
50.1b ±10.2 (10) Kruskal–Wallis/Student– Newman–Keuls
0.0b ±0.0 (10) Kruskal–Wallis/Student– Newman–Keuls
Thymol 1.0% Thymol 1.5% Thymol 2.0% Amitraz Statistical test
Means followed by the same letters in the same column do not differ statistically at a significance level of 5% n, sample size=number of engorged females per treatment;
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Table 33 Pre-oviposition Pre-oviposition period, period,hatching hatchingpercentage, percentage, eggegg production production thymol, index (EPI), underand laboratory nutritionalconditions index (NI)(27de ± 1°C engorged e UR>80 females ± 10%) de Rhipicephalus index (EPI), and sanguineus nutritional treated index with(NI) different de engorged concentrations femalesof de thymol, (Advanced under laboratory Laboratory conditions of Zoology, (27±1°C UFJF)e UR>80±10%) (Advanced Laboratory of Zoology, Rhipicephalus sanguineus UFJF) treated with different concentrations of Concentrations of thymol
Pre-oviposition period (days)
Hatching percentage (% H)
Egg production index (EPI)
Nutritional index (NI)
Water+DMSO
4.1ª±0.3 (9) 3.9ª±0.5 (16) 4.0ª±0.4
89.4ª±27.6 (9) 93.8ª±6.7 (16) 86.0ª±26.4
55.6ª±6.7 (9) 49.34a ±7.7 (16) 50.33a ±13.9
65.7ª±9.7 (9) 63.7a ±7.9 (16) 62.1a ±17.1
(16) 4.0ª±0.2 (13) – Anova
(14) 86.3ª±20.7 (11) – Kruskal–Wallis
(16) 51.73a ±4.8 (13) – Kruskal–Wallis/Student– Newman–Keuls
(16) 63.9a ±4.5 (13) – Kruskal–Wallis/Student– Newman–Keuls
Thymol 1.0% Thymol 1.5% Thymol 2.0% Amitraz* Statistical test
Means followed by the same letters in the same column do not differ statistically at a significance level of 5% n, sample size=number of engorged females per treatment; (–) the statistical analysis was not performed due to the insufficient sample size
was obtained in thymol concentration of 2%. The results obtained by Daemon et al. (2009) and in the present work, for engorged larvae and nymphs of R. sanguineus, respectively, make it evident that thymol concentration of 2% had 100% efficacy, a fact that was not observed in engorged females. Different works demonstrate that immature stages are more susceptible to the acaricide effect. Bittencourt (1987) evaluated the efficacy of some synthetic pyrethroids on different stages of Amblyomma cajennense and concluded that adults were more resistant than the immature ones. It was also observed by Sant'anna et al. (2002), who reported that R. sanguineus adults were more resistant to different concentrations of alphamethrin, comparing to larvae and nymphs. In relation to vegetable origin products, Ribeiro et al. (2008) evaluated the acaricide effect of hexane extract of Calea serrata and
Fig. 1 Control percentage of engorged females of Rhipicephalus sanguineus treated with different concentrations of thymol, under laboratory conditions at 27±1°C and UR >80% (Advanced Laboratory of Zoology, UFJF)
attested that B. microplus larvae are more susceptible than engorged females. R. sanguineus is a tick of three hosts that shows nidicolae habit, and thus, all the processes related to its non-parasitary phases (ecdyses, posture, and egg incubation) happen in cracks or holes present on walls or roofs where hosts live (Labruna 2004; Dantas-Torres 2008). For this reason, in order to be controlled, it is necessary to perform it with applications of topic acaricides in animals and acaricides of aspersion in the environment where the host is located (Labruna 2004; Paz et al. 2008). In this context, thymol represents a promising agent for the control of this tick, since emulsion-concentrated thymol could reduce the number of applications of synthetic acaricides, minimizing the amount of chemical products in the environment and the risk of animal and human intoxication. Besides the advantages already mentioned, in the USA, thymol is recognized as a safe component by the Food and Drug Administration and by the Environmental Protection Agency, and it was approved for applications in field plantations of comestible products (Ji et al. 2005). The utilization of thymol may also represent waste reduction with the acquisition of synthetic pesticides, a fact attested by Ji et al. (2005) in relation to the use of this monoterpene against the bacteria Ralstonia solanacearum, an agent that causes tomato bacterial wilt in field plantations. A recent note of the Food and Agriculture Organization classified Brazil as the third largest user of pesticides and, as a coincidence or not, the third on cancer mortality (Ponte 1999). Hence, it becomes a matter of extreme importance to develop pest control strategies in order to create new alternatives for the pest control, reducing the application of
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synthetic chemical pesticides. Therefore, it is important to carry out new studies that simulate field conditions aiming to accomplish a more precise definition of the potential of this vegetable origin product for the control of this and other tick species.
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