Parasitol Res (2009) 105:10931097 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 Mrcio 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 271C and UR >8010%, 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 (p <0.05) in the parameters of weight alteration, egg mass weight, pre-oviposition period, hatching percentage, egg production index and nutritional index; however, it affected the engorged females final weight in all treatments (p >0.05). 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: -caiosat@gmail.com

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 (60C) in aqueous dimethylsulfoxide (DMSO, 1%). Experiment 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 (AmitrazTriatox, 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 271C and UR >8010% 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 mass100/initial weight; nutritional index (NI), obtained according to the formula proposed by Bennett (1974), where NI=egg mass100/(initial weight final weight); estimate of reproduction (ER), obtained by the formula, (PP/PI)%EC20.000 (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. 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 271C 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: Mortality% dead nymphs 100 Total tick

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 (p <0.05). In the case of nonparametric distribution, the values were compared through nonparametric tests of KruskalWallis and StudentNewmanKeuls (p <0.05).

Results The results for each test with nymphs are demonstrated in Table 1. The group treated with thymol concentration of 0.25% did not show any significant differences (p >0.05) when compared to the control group, whereas the mortality rate was 100% in other concentrations, differing significantly (p <0.01) from the control group and being statistically similar to the positive control group (p >0.05). The results for the test with the engorged females are presented in Tables 2 and 3 and in Fig. 1. The initial weight values for females of different groups did not show any significant differences (p >0.05) (Table 2). The values

Experiment 2 In order to perform the test, 80 females were used, which were weighted and separated in three groups of 20 (each

Parasitol Res (2009) 105:10931097 Table 1 Mean mortality of engorged nymphs of Rhipicephalus sanguineus treated with different concentrations of thymol under laboratory conditions (27 1C and UR >80 10%) (Advanced Laboratory of Zoology, UFJF) Concentrations of thymol Water+DMSO Thymol 0.25% Thymol 0.50% Thymol 1.0% Thymol 1.5% Thymol 2.0% Amitraz Mortality percentage (X SD) 0.0a 0.0 n =(10) 0.0a 0.0 n =(10) 100.0b 0.0 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)

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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 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

Means followed by the same letters in the same column do not differ statistically at a significance level of 5%. (KruskalWallis/Student NewmanKeuls) 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).

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 (271C e UR>8010%) (Advanced Laboratory of Zoology, UFJF) Concentrations of thymol Water+DMSO Thymol 1.0% Thymol 1.5% Thymol 2.0% Amitraz Statistical test Initial weight (mg) Final weight (mg) Alteration weight (mg) Egg mass weight (mg)

131.029.1 (9) 131.424.9 (20) 131.630.1 (20) 133.925.4 (20) 135.725.6 (10) ANOVA

19.67.1 (9) 43.7b 34.0 (20) 42.2b 41.5 (20) 60.2bc 46.3 (20) 85.5c 24.0 (10) KruskalWallis/Student NewmanKeuls

111.325.6 (9) 87.641.7 (20) 89.239.1 (20) 73.6ab 53.1 (20) 50.1b 10.2 (10) KruskalWallis/Student NewmanKeuls

72.114.3 (9) 53.732.3 (20) 55.933.0 (20) 47.438.4 (20) 0.0b 0.0 (10) KruskalWallis/Student NewmanKeuls

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 3 3 Pre-oviposition Pre-oviposition period, period,hatching hatching percentage, percentage, egg egg production production thymol, index (EPI), underand laboratory nutritional conditions index (NI) (27 de 1C engorged e UR>80 females 10%) de Rhipicephalus index (EPI), and sanguineus nutritional treated index with (NI) different de engorged concentrations females of de thymol, (Advanced under laboratory Laboratory conditions of Zoology, (271C UFJF)e UR>8010%) (Advanced Laboratory of Zoology, Rhipicephalus sanguineus UFJF) treated with different concentrations of Concentrations of thymol Water+DMSO Thymol 1.0% Thymol 1.5% Thymol 2.0% Amitraz* Statistical test Pre-oviposition period (days) Hatching percentage (% H) Egg production index (EPI) Nutritional index (NI)

4.10.3 (9) 3.90.5 (16) 4.00.4 (16) 4.00.2 (13) Anova

89.427.6 (9) 93.86.7 (16) 86.026.4 (14) 86.320.7 (11) KruskalWallis

55.66.7 (9) 49.34a 7.7 (16) 50.33a 13.9 (16) 51.73a 4.8 (13) KruskalWallis/Student NewmanKeuls

65.79.7 (9) 63.7a 7.9 (16) 62.1a 17.1 (16) 63.9a 4.5 (13) KruskalWallis/Student NewmanKeuls

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 271C 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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1097 Imdorf A, Kilchenman V, Bogdanov S (1995) Toxizit von thymol; campher, menthol and eucaliptol auf Varroa jacobsoni oud ind Apis mellifera L. in labortest. Apidologie 26:2731 Ji P, Momol MT, Olson SM, Pradhanang PM, Jones JB (2005) Evaluation of thymol as biofumigant for control of bacterial wilt of tomato under field conditions. Plant Dis 89:497500 Labruna MB (2004) Biologica-ecologia de Rhipicephalus sanguineus (Acari: Ixodidae). Rev Bras Parasitol Vet 13:123124 Louly CCB, Fonseca IN, Oliveira VF, Borges LMF (2006) Ocorrncia de Rhipicephalus sanguineus em trabalhadores de clnicas veterinrias e canis, no municpio de Goinia, GO. Cienc Anim Bras 7:103106 Martins JRS, Furlong J, Leite RC (2006) Controle de carrapatos. p.145153. In: Barros-Battesti DMB, Arzua M, Bechara GH (eds) Carrapatos de importncia mdico-veterinria da Regio Neotropical. Um guia ilustrado para a identificao de espcies. Instituto Butantan, So Paulo, p 223 Matsumoto K, Brouqui P, Raoult D, Parola P (2005) Experimental infection models of ticks of the Rhipicephalus sanguineus group with Rickettsia conori. Vect Borne Zoonotic Dis 5:363372 Mansour SA, Messehea SS, El-Gengaihi SE (2000) Botanical biocides. 4. Mosquitocidal activity of certain Thymus capitatus constituents. J Nat Toxins 9:4962 Miller RJ, George JE, Guerreiro F, Carpenter L, Welch JB (2001) Characterization of acaricide resistance in Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) collected from the Corozal Army Veterinary Quarantine Center, Panama. J Med Entomol 38:293302 Neitz WO, Boughton F, Walters HS (1971) Laboratory investigations on the life caroo paralysis tick Ixodes rubidicundus (Neumann, 1904). J Vet Res 38:215224 Novelino AMS, Daemon E, Soares GLG (2007) Evaluation of acaricide effect of thymol, menthol, salycilic acid and methyl salycilate on Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae) larvae. Parasitol Res 101:809811 Paster N, Menasherov M, Ravid U, Juven B (1995) Antifungal activity of oregano and thyme essential oils applied as fumigants against fungi attacking stored grain. J. Food Prot 58:8185 Paz GF, Leite RC, Oliveira PR (2008) Controle de Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) no canil da Escola de Veterinria da UFMG, Belo Horizonte, Minas Gerais, Brasil. Rev Bras Parasitol Vet 17:4144 Ponte JJ (1999) Cartilha da manipueira, uso do composto como insumo agrcola. SECITECE, Fortaleza p 53 Ribeiro VLS, Avancini C, Gonalves K, Toigo E, Von Poser GL (2008) Investigation of the acaricidal activity of Calea serrata (Asteraceae) on Boophilus microplus and Rhipicephalus sanguineus. Vet Parasitol 151:351354 Rozental T, Bustamante MC, Amorim M, Serra-Freire NM, Lemos ER (2002) Evidence of spotted fever group rickettsiae in state of Rio de Janeiro, Brazil. Rev Inst Med Trop So Paulo 44:155158 Sant'anna FB, Torres FO, Martins IVF, Correia TR, Fernandes JI, Freitas IF, Scott FB, Grisi L (2002) Eficcia do piretride sinttico alfametrina no controle de Rhipicephalus sanguineus em ces. Parasitol Latinoam 57:3033 Sousa R, Bacellar F (2004) Morbi-Mortalidade por Rickettsia conorii em Portugal. Rev Bras Parasitol Vet 13:180184

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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