Parasitol Res (2009) 105:495–497 DOI 10.1007/s00436-009-1426-9
ORIGINAL PAPER
Evaluation of the acaricide activity of thymol on engorged and unengorged larvae of Rhipicephalus sanguineus (Latreille, 1808) (Acari: Ixodidae) Erik Daemon & Caio Márcio de Oliveira Monteiro & Leonardo dos Santos Rosa & Mateus Aparecido Clemente & Alessandro Arcoverde
Received: 14 March 2009 / Accepted: 18 March 2009 / Published online: 28 March 2009 # Springer-Verlag 2009
Abstract The present work was aimed at evaluating the in vitro efficacy of thymol on engorged and unengorged larvae of Rhipicephalus sanguineus. In order to perform the study, unengorged larvae were interleaved on filter paper (2×2 cm) recently sprayed with thymol in the following concentrations: 0.25%, 0.5%, 1.0%, 1.5%, and 2.0%. For each concentration, ten repetitions of 50 larvae each were performed. A control group was established (water + DMSO) together with a positive control group (Amitraz), with ten repetitions each. Mortality was evaluated after 24 h. For the test with engorged larvae, the same thymol concentrations were used with the larvae being immersed for 5 min and mortality evaluated after 15 days. The values found for thymol efficacy for unengorged larvae were of 21.3%, 17.4%, 19.1%, 32.1%, 37.7% and for engorged larvae 3.0%, 0.0%, 3.0%, 97.0%, and 100.0% in concentrations of 0.25%, 0.5%, 1.0%, 1.5%, and 2.0% respectively. We have concluded that thymol had a deleterious effect on larvae of R. sanguineus, especially on engorged larvae for which 100% mortality was found in higher concentration.
Introduction Tick control is normally carried out by means of synthetic acaricide products, which, in spite of significantly contrib-
E. Daemon (*) : C. M. de Oliveira Monteiro : L. dos Santos Rosa : M. Aparecido Clemente : A. Arcoverde Universidade Federal de Juiz de Fora—UFJF, Juiz de Fora, Brazil e-mail:
[email protected] e-mail:
[email protected]
uting to the control of these arthropods, generate environment-unfriendly waste and, in case of animals, food chemical traces (Miller et al. 2001; Martins et al. 2006). Therefore, new alternatives have been sought in order to minimize the problem and because of that, vegetable products have gained more and more ground, as they present lower risk of environment contamination and slower resistance development rate (Baladrin et al. 1985; Chagas 2004) Thymol (= 5meil-2-isopropil-1-fenol) is a volatile, refringent monoterpenoid, found in plants of the Lamiacea and Apiacea families. The insecticide, molluscicidal, and acaricidal effects of thymol have already become evident for a number of species, by means of using different formulations and ways of application suitable for each target species (Indorf et al. 1995; Mansour et al. 2000; Ferreira 2005). However, the acaricide effect of thymol on engorged larvae of Boophilus microplus (Canestrini, 1887) has only recently been demonstrated by Novelino et al. (2007). In order to enrich the studies of this activity in other tick species, the present work evaluated the acaricide effect of thymol on engorged and unengorged larvae of the brown dog tick Rhipicephalus sanguineus (Latreille 1808) (Acari, Ixodidae), an important vector species of pathogenic agents for both dogs and men (Matsumoto et al. 2005; Demma et al. 2005; Dantas-Torres 2008).
Material and methods The experiments were performed at the Advanced Laboratory of Zoology of the Department of Zoology of the Federal University of Juiz de Fora, Minas Gerais, Brazil.
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Tick collecting and maintenance Engorged females were collected in dogs infested naturally, without recent contact with acaricide products. After the collection, the females were cleaned with a soft paintbrush and placed on Petri’s plates, which were kept in a climatized room at 27ºC and humidity levels over 80%. Every 3 days after the beginning of oviposition, the eggs were gathered and weighed in 100-mg packets placed in disposable plastic syringes with distal edges cutoff and isolated with hydrophilic cotton. The syringes were kept under the same conditions as the females. Around 15 days after the hatching, part of the larvae underwent treatment, and the rest were employed in the artificial infestation of rabbits, according to the technique developed by Neitz et al. (1971) for obtaining engorged larvae. After this process, the larvae underwent the same treatments as the unengorged larvae. Due to their low water solubility, thymol solutions were emulsified under heating (60ºC) in aqueous dimethylsulfoxide (DMSO 1%). Experiments Seven groups were formed: positive control (aqueous solution of DMSO at 1%), negative control with Amitraz (Triatox®, Coopers), and aqueous solutions of DMSO at 1% containing 0.25%, 0.5%, 1.0%, 1.5%, and 2.0% of thymol. For each treatment, ten repetitions were performed. In order to perform the test for unengorged larvae, a larval packet technique was employed, as proposed by Stone and Haydock (1962) and adapted by Leite (1988), by which approximately 100 larvae were interleaved with filter paper (2×2 cm) and sprayed with the concentrations studied. After that, the envelopes were sealed and kept in a climatized room (27±1ºC and UR>80±10%). After 24 h, a mortality evaluation was performed with the help of a vacuum pump with a pipet attached to a rubber tube. The mortality rate for these treatments was obtained according to the following formula: dead larvae Mortality ð%Þ ¼ 100 total of larvae In the second test, the engorged larvae were immersed in the thymol solutions mentioned above for 5 min, in an adaptation of the technique of immersion of engorged females described by Drummond et al. (1973). After the immersion period, the larvae were placed into test tubes properly labeled, sealed with hydrophilic cotton, and kept in a climatized room at 27±1°C and UR>80± 10%. After 15 days, mortality evaluation was performed.
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The mortality rate for the treatments was obtained according to the formula mentioned above.
Statistical analysis The percentage values were transformed into 2√arcsen x. The median values of each treatment were analyzed by analysis of variance and Tukey test (P0.01) in the groups of 1.5% and 2%, compared to the others and the control group (water +
Table 1 Mean mortality of engorged and unengorged larvae of Rhipicephalus sanguineus treated with different concentrations of thymol under laboratory conditions (27±1°C and UR>80±10%) Treatment
water + DMSOa Amitraz 0.25* 0.50* 1.0* 1.5* 2.0*
Mortality Percentage (X±SD) Unengorged larvae
Engorged larvae
7.9a ±5.0 97.8d ±3.8 21.3b ±16.7 17.4b ±7.2 19.1b ±1.9 32.1c ±7.9 37.7c ±13.4
3.0a ±6.7 100.0b ±0.0 3.0a ±4.8 0.0a ±0.0 3.0a ±4.8 97.0b ±6.7 100.0b ±0.0
Mean values followed by identical letters in the same column do not differ statistically at a significance level of 5% a
Dimetilsulphoxide
b
Concentration of thymol in each treatment
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DMSO). However, contrary to what was found for ungorged larvae, mortality rates were extremely high, reaching 100% in the group undergoing concentrations of 2%, the same value found for the positive control group.
Discussion As it is known, the several tick species present different levels of sensibility to, acaricide products, noticeably those of natural origin. Therefore, the results found for unengorged larvae in the present work confirm such information, once a water solution of 1% thymol was enough to kill 100% of the exposed larvae of B. microplus (Novelino et al. 2007). For R. sanguineus, however, not even twice as much this concentration was enough to perform mortality levels which could make thymol an effective acaricide product at this stage of the cycle. Due to the lack of sound information about the action of thymol on ticks, eventual explanations about this discrepancy have a speculative meaning. A possible explanation would be the known fact that R. sanguineus is a species originating from the African continent, adapted to xeric conditions (Labruna 2004; Dantas-Torres 2008), which could lead to decreased cuticular permeability, therefore stopping the action of thymol. By contrast, the acaricide action of thymol on the engorged larvae can be considered to be satisfactory, bearing in mind that the mortality levels were similar to the positive control group. According to what has been exposed, it is possible that the extension of the cuticle, stimulated by engorgement, has increased its permeability, thus creating suitable conditions for the acaricide action of the product. According to Odhiambo (1982), the cuticle gets thicker at the beginning of the blood meal, but as engorgement develops, it gets thinner. Taking into account that the control of R. sanguineus includes measures aimed at reducing the infestation in dog runs (Labruna and Pereira 2001; Labruna 2004; Paz et al. 2008), the thymol can be considered a promising supporting agent for the control of R. sanguineus, since cracks and crevices are the places where moulting and oviposition of this species occur (Labruna 2004). The results found for this work, in contrast to those obtained by Novelino et al. (2007), clearly point to the need for a better understanding of the action of thymol on ticks. Such understanding could lead to the search of solutions which would allow its increased acaricide action. On the other hand, it is necessary to evaluate the acaricide action of thymol on the different species (and stages of the lifecycle of ticks), so as to reach a precise definition of the potentialities of the product.
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