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Experimental Parasitology 129 (2011) 175–178

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Experimental Parasitology

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Acaricidal activity of the essential oil from riparia () on the cattle tick Rhipicephalus (Boophilus) microplus (Acari; Ixodidae)

Zilda Cristiani Gazim a, Izabel Galhardo Demarchi b, Maria Valdrinez Campana Lonardoni b, Ana Carolina L. Amorim c, Ana Maria C. Hovell c, Claudia Moraes Rezende c, Gilberto Alves Ferreira d, ⇑ Edson Luiz de Lima d, Fábio Antunes de Cosmo d, Diogenes Aparício Garcia Cortez a,

a Departamento de Farmácia, Universidade Estadual de Maringá, Av. Colombo s/no, Maringá, PR, Brazil b Departamento de Análises Clínica, Universidade Estadual de Maringá, Av. Colombo s/no, Maringá, PR, Brazil c Instituto de Química, Universidade Federal do Rio de Janeiro, Av. Brigadeiro Trompowski s/no, Rio de Janeiro, Brazil d Departamento de Farmácia, Universidade Paranaense, Unipar, Praça Mascarenhas de Moraes s/no, Umuarama, Paraná, Brazil

article info abstract

Article history: (Lamiaceae) is a well-known herbal medicine with a variety of useful properties, Received 10 November 2010 including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of T. Received in revised form 20 June 2011 riparia essential oil (EO) against engorged females of Rhipicephalus (Boophilus) microplus (Acari; Ixodidae). Accepted 28 June 2011 For this purpose, nine serial concentrations (12.50%, 6.25%, 3.75%, 1.80%, 0.90%, 0.45%, 0.22%, 0.11%, and Available online 5 July 2011 0.056% w/v) of T. riparia were used for the adult immersion test (AIT). For the larval packet test (LPT), we used 14 serial concentrations (100.00%, 50.00%, 25.00%, 12.50%, 6.25%, 3.65%, 1.82%, 0.91%, 0.45%, 0.228%, Keywords: 0.114%, 0.057%, 0.028%, and 0.014% w/v). The results for AIT showed 100.00% and 2.05% mortality, 19.00 Tetradenia riparia and 90.20% for the total number of eggs, egg-laying inhibition of 0.00% and 90.20%, hatchability inhibition Acaricide Ixodidae of 0.00% and 70.23%, and product effectiveness of 100.00% and 2.89%, respectively. The AIT indicated that Tick the LC50 and LC99.9, calculated using the Probit test, were for mortality (%) 0.534 g/mL (0.436–0.632) and Rhipicephalus (Boophilus) microplus 1.552 g/mL (1.183–1.92); for total number of eggs were 0.449 g/mL (0.339–0.558) and 1.76 g/mL (1.27–2.248); and for hatchability inhibition were 0.114 g/mL (0.0–0.31) and 2.462 g/mL (1.501–3.422), respectively. Larvae between 14 and 21 days old were fasted and placed in each envelope. Bioassays were performed at 27° ±1°C, RH P 80%. Larval mortality was observed 24 h after treatment and showed

10.60–100% mortality in the LPT bioassay. The LPT showed that the LC50 and LC99.9 were 1.222 g/mL (0.655–1.788) and 11.382 g/mL (7.84–14.91), respectively. A positive correlation between T. riparia EO concentration and tick control, was observed by the strong acaricidal effects against R. (B.) microplus, and the mortality rate of ticks was dose-dependent. Our results showed that T. riparia is a promising candidate as an acaricide against resistant strains of R. (B.) microplus. Crown Copyright Ó 2011 Published by Elsevier Inc. All rights reserved.

1. Introduction in society and government, by harming the animals themselves and humans who consume the products from these animals In Brazil, the main tick species that undermines the productivity (Chagas et al., 2003). Each time that ticks survive an application of cattle raising is Rhipicephalus (Boophilus) microplus. The damage of insecticide, they transmit genetic information to later genera- caused by this tick to South American cattle exceeds two billion tions about how to survive that product (Furlong et al., 2004). dollars a year (Grisi et al., 2002). Primarily by biting, it affects The use of extracts in tick control has also been the focus the production of meat and milk but the tick also injects toxins into of extensive research (Martinez-Velazquez et al., 2010). Tetradenia the host and transmits infectious agents (Olivo et al., 2008). The riparia (Hochstetter) Codd, a member of the family Lamiaceae, is a control of cattle ticks is usually done using conventional chemicals shrub common throughout Africa. In , it is one of the including synthetic pyrethroids (SP), organophosphates (OP), and most popular herbs and medicinal (Van Puyvelde and De amitraz (Am). These cause the rapid development of resistance to Kimpe, 1998). For decades, T. riparia has been the subject of the active principle. Further, their use has caused great concern research to isolate and identify the active compounds present in extracts from its leaves. Several studies have been conducted to evaluate the biological activities of T. riparia: as larvicide (Weaver ⇑ Corresponding author. et al., 1992); insecticide (Weaver et al., 1994); antimalarial E-mail address: [email protected] (D.A.G. Cortez). (Campbell et al., 1997) and repellent effects on Anopheles gambiae

0014-4894/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.06.011 176 Z.C. Gazim et al. / Experimental Parasitology 129 (2011) 175–178

(Omolo et al., 2004). To date, no studies reported in the literature To implement the LPT test, larvae were obtained from en- on the acaricidal activity of this plant. Thus the present experiment gorged females of R. (B.) microplus, and allowed to rest unfed was to evaluate the acaricidal activity of the essential oil from fresh for 14–21 days following hatchability prior to their use. The lar- leaves of T. riparia against the acari of R. (B.) microplus. vae were exposed to test solutions in filter-paper envelopes (2 2 cm), containing micropores to allow better ventilation (Fer- 2. Materials and methods nandes et al., 2008). The following treatments were used: (1) enve- lopes of dry filter paper; (2) envelopes of filter paper moistened 2.1. Plant material with a negative control (solution of 2% Tween 80 in distilled water); (3) positive controls consisting of water with 0.125% Colos- Ò Plant material (leaves) of T. riparia was collected in a medicinal- so Pulverização (cypermethrin 0.25; chlorpyrifos 0.25 and citro- plant garden at the Universidade Paranaense (Unipar), northwest- nellal 0.01 g/mL) and (4) envelopes moistened with 2 mL of each ern Paraná State, Brazil. A voucher specimen was authenticated concentration of essential oil of T. riparia to be tested. After each and deposited at the herbarium of the University Educational envelope was treated and larval ticks inserted, the opening was Paranaense (HEUP), under number 2502. To obtain the essential folded over (10 mm) and re-sealed with a metallic clip, with its oil of T. riparia, fresh leaves were collected between 06:30 and identification mark (solution and concentration tested) on the out- 08:00 a.m. during summer, between 21/12/2008 and 21/03/2009, side. The packets were placed in the BOD incubator at a tempera- and hydrodistilled for 3 h using a modified Clevenger-type appara- ture of 27–28 °C and 85–95% relative humidity for 24 h. The tus. The distilled oils were collected and dried over anhydrous envelopes were then opened and inspected, using a stereomicro- sodium sulfate and stored in a freezer (Gazim et al., 2010). scope, to record the number of live and dead larvae, and any toxi- cological effects observed. Each treatment contained three 2.2. Ticks replicates:

About 300 engorged females of R. (B.) microplus were collected Corrected percent mortality from naturally infested cattle on farms in Tapejara city, Paraná, % test mortality % control mortality ¼ 100 which had suspended the use of acaricidal treatment for at least 100 % control mortality 45 days prior to the study. The females were used in the AIT or incubated at 27–28 °C and 70–80% relative humidity for 2 weeks until they laid eggs. 2.4. Statistical analyses 2.3. Bioassays (AIT and LPT) The experimental design was completely randomized. The data were processed and subjected to analysis of variance (ANOVA), and For the acaricidal test (AIT), we made serial dilutions (12.50%, differences between means were determined by Tukey test at 5% 6.25%, 3.75%, 1.80%, 0.90%, 0.45%, 0.22%, 0.11%, and 0.055% w/v) significance level. Lethal concentrations (LC) to kill 50% and 99% of essential oil of T. riparia. For the larval packet test (LPT) the con- of larvae and their respective 95% confidence intervals (CI) were centrations used were 100.00%, 25.00%, 12.50%, 6.25%, 3.65%, calculated by probit analysis (software R version 11.2). 1.82%, 0.91%, 0.45%, 0.228%, 0.114%, 0.057%, 0.028%, and 0.014% w/v. In both tests, the essential oil was diluted in an aqueous solution containing 2.0% of an emulsifying agent (Tween 80 v/v), 3. Results described by Farias et al. (2007). The AIT was described by Drummond et al. (1976), where groups of 30 engorged females The essential oil of T. riparia was analyzed by Gazim et al. were weighed and immersed for 5 min in the respective dilutions (2010), and 31 compounds, comprising 97.17% of the oil, were (10 mL) in a 50-mL beaker which was gently agitated (three times) identified. The predominant class in this oil was oxygenated at room temperature. The emulsifying solution (2% Tween) was sesquiterpenes: 14-hydroxy-9-epi-cariophyllene (18.03%), cis- used as the negative control (Farias et al., 2007; Rosado-Aguilar muurolol-5-en-4-a-ol (11.73%), ledol (7.18%), and a-cadinol et al., 2010). Water with 0.125% Colosso PulverizaçãoÒ (cyper- (4.90%), followed by monoterpene hydrocarbons: limonene methrin 0.25; chlorpyrifos 0.25 and citronellal 0.01 g/mL) was used (3.69%), and oxygenated: fenchone (12.87%). as a positive control. Ticks were removed from the solutions, dried, The percentages obtained for in vitro efficacy of the oil of T. ripa- and each group placed in a separate Petri dish. The Petri dishes ria against R. (B.) microplus are shown in Table 1. In the AIT, the effi- were incubated at 27–28 °C and 70–80% relative humidity. After cacy of treatment against engorged females was assessed by 14 days, the number of females laying eggs was recorded, and measuring mortality of gravid females, total number of eggs, egg the eggs were collected, weighed, and observed. The eggs were weight, percentage of hatchability, and product efficiency (Table placed in glass tubes, incubated at 27–28 °C and 70–80% relative 1). Table 2 shows the percent mortality of R. (B.) microplus larvae humidity, and after 21 days the percentage of hatched eggs was exposed to different concentrations of T. riparia essential oil calculated. The effectiveness of the product (PE) was calculated through the larval packet test (LPT). In dilutions of 100%, 50%, using the mathematical formulas below, described by Drummond and 25% the oil killed 100% of the larvae, indicating a maximum et al. (1973). Each treatment contained three replicates: efficiency. In dilutions from 12.5% to 0.014%, the mortality rate of larvae was high, ranging from 97.6% to 10.60%, respectively, with Reproduction estimated ðREÞ no significant differences between them. However, all these results egg weight % hatchability 20:000 differed significantly from the negative control (8.00%). The LC ¼ 50 weight of females and LC99.9 (Table 3) calculated using the Probit test, were for% mor-

⁄ tality 0.534 g/mL (0.436–0.632) and 1.552 g/mL (1.183–1.92), for Constant indicating the number of eggs present in 1 g of egg laying. total number of eggs 0.449 g/mL (0.339–0.558) and 1.76 g/mL Effectiveness of the product ðPEÞ (1.27–2.248), and the hatchability inhibition was 0.114 g/mL (0.0–0.31) and 2.462 g/mL (1.501–3.422), respectively. The larval RE ðcontrol groupÞRE ðtreated groupÞ ¼ 100 mortality (LPT) was 1.222 g/mL (0.655–1.788) and 11.382 g/mL RE ðcontrol groupÞ (7.84–14.91), respectively. Z.C. Gazim et al. / Experimental Parasitology 129 (2011) 175–178 177

Table 1 Mean ± (standard error) mortality of gravid females, total number of eggs, egg weight, percentage of hatchability and product efficiency in engorged females of Rhipicephalus (Boophilus) microplus after immersion in different dilutions of essential oil T. riparia.

T. riparia essential oil (% w/v) % Mortality Total number of eggs Egg weight (g) Hatchability (%) Effectiveness of the product (%) C1 100 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100 ± 0.00 12.5 100 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100 ± 0.00 6.25 100 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100 ± 0.00 3.75 100 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100 ± 0.00 1.80 100 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100 ± 0.00 0.90 76.68 ± 15.27a 19.00 ± 2.22a 0.068 ± 0.010a 34.87 ± 6.23a 64.27 ± 10.66a 0.45 53.33 ± 15.27 b 35.87 ± 3.00b 0.092 ± 0.010a 24.13 ± 1.68a 68.28 ± 5.63a 0.22 23.33 ± 5.77c 60.27 ± 2.93c 0.066 ± 0.010a 45.47 ± 2.08a 45.63 ± 7.25b 0.11 10.00 ± 10.00d 80.12 ± 7.30d 0.075 ± 0.010a 59.47 ± 2.79b 25.72 ± 10.01c 0.056 2.05 ± 2.30e 90.20 ± 9.42e 0.095 ± 0.010a 70.23 ± 2.25c 2.89 ± 3.01d C2 1.10 ± 1.90d 91.30 ± 5.08e 0.075 ± 0.010a 73.87 ± 2.24c 0.00 ± 0.00

C1: positive control (water with 0.125% of Colosso PulverizaçãoÒ); C2: negative control (water 2% Tween 80). Values in the same column with different subscript are significantly different; analyzed by Tukey test at 5% probability (p < 0.05).

Table 2 Percent mortality of Rhipicephalus (Boophilus) microplus stages of R. (B.) microplus, and the mortality rate of the ticks was larvae exposed to different concentrations of T. riparia dose-dependent. Many essential oils and their components, mainly essential oil. monoterpenoids and sesquiterpenoids, show repellent, chemoster- T. riparia essential % Average mortality ilant, antifeeding, and biocidal activities against the cattle tick R. oil (% w/v) (B.) microplus (Facey et al., 2005). However, the chemical constitu- C1 99.6 ± 1.22a ents of essential oils from other plant species differ from those 100.00 100.00 ± 0.00 found in the essential oil of T. riparia. For example, the oil of T. ripa- 50.00 100.00 ± 0.00 ria killed 100% of gravid female ticks at concentrations from 1.8% to 25.00 100.00 ± 0.00 12.5%. Similarly, the studies of Martins (2006) with essential oil of 12.50 97.60 ± 1.21a Cymbopogon winterianus (citronella or Java grass), which is rich in 6.25 96.30 ± 1.20a 3.65 94.60 ± 1.20a monoterpenes (41.15% citronellal), showed 100% mortality at con- 1.82 74.60 ± 1.20b centrations from 12.5% to 50.0%. Chagas et al. (2002) investigated 0.91 62.30 ± 1.19b the action of monoterpene-rich essential oil from three species of b 0.45 58.60 ± 1.17 Eucalyptus: Eucalyptus citriodora (94.9% citronellal), Eucalyptus glob- 0.228 56.60 ± 1.16b 0.114 46.60 ± 1.16c ulus (9.93% a-pinene), and Eucalyptus staigeriana (24.78% DL-limo- 0.057 44.60 ± 1.16c nene), in five different concentrations, against gravid female 0.028 24.60 ± 1.16d ticks. The results showed that E. citriodora, E. globulus and E. staige- e 0.014 10.60 ± 1.16 riana killed 100% of females at mean concentrations of 17.50%, 15% C2 8.00 ± 0.76e and 12.5%, respectively. C1: positive control (eater with 0.125% of Colosso Regarding the inhibitory action of T. riparia essential oil on ovi- Ò Pulverização ); C2: negative control (water 2% Tween position, there was a 100% reduction of egg laying with oil concen- 80). trations from 1.80% to 12.5%. This effect was also evaluated by Values in the same column with different subscript are significantly different; analyzed by Tukey test at 5% Olivo et al. (2008), who investigated the effect of the essential oil probability (p < 0.05). from C. nardus containing high levels of monoterpenes (50.0% citro- nellal), and observed 90% inhibition of oviposition at concentra- tions of 100%, 50%, 25% and 10%. Also, Ribeiro et al. (2010) found Table 3 that essential oil from H. ringens, which has a high content of oxy-

Lethal concentration LC99.9 and LC50 of T. riparia essential oil for R. (B.) microplus and genated monoterpenes (86.0% Pulegone), inhibited egg laying by respective confidence intervals, calculated starting from the interpolation of the 76.4% and 48.2% at concentrations of 5.0% and 2.5%, respectively. acaricidal test (AIT) mortality of gravid females (%), total number of eggs and Another parameter evaluated was the influence of T. riparia essen- hatchability results, and larval packet test (LPT) by Probit analysis. tial oil on the percentage of hatching of larvae. The results showed LC50 (g/mL) (CI) LC99.9 (g/mL) (CI) 100% inhibition of hatching at concentrations ranging from 1.8% to Acaricidal test (AIT) 12.5%. Ferrarini et al. (2008) evaluated the effect of limonene Mortality of gravid 0.534 (0.436–0.632) 1.552 (1.183–1.92) (monoterpene hydrocarbons) in inhibiting egg hatching of R. (B.) females (%) microplus, at concentrations from 0.25% to 1.0%, and obtained Total number of eggs 0.449 (0.339–0.558) 1.76 (1.27–2.248) Hatchability (%) 0.114 (0.0–0.31) 2.462 (1.501–3.422) 91% to 100% inhibition. Taken together, the results showed that oil has a significant potential to control the larvae of R. (B.) micro- Larval packet test (LPT) Mortality larvae (%) 1.222 (0.655–1.788) 11.382 (7.84–14.91) plus. Ferraz et al. (2010) observed that for the essential oil of Piper amalago, which contained high levels of monoterpenes (20.52% LC50: lethal concentration 50%; LC99.9: lethal concentration 99.9%; CI: confidence limonene) and sesquiterpenes (11.18% zingiberene), the most intervals. active concentration with LC50 was 0.23%. Ribeiro et al. (2008) evaluated the larvicidal effect of Drimys brasiliensis essential oil, which contained predominantly sesquiterpenes (30.4% cycloco- 4. Discussion lorenone), on the larvae of R. (B.) microplus. One hundred percent of the larvae were killed at concentrations of 2.5%, 1.25% and The analyses of the essential oil from T. riparia by GC–MS iden- 0.625%. Chagas et al. (2002) also observed that the essential oil tified a mixture of sesquiterpenes (50.30%) and monoterpenes of E. staigeriana killed 100% of the larvae at a concentration of (19.52%). The essential oil showed intense activity against different 10%, and E. globulus had the same effect at a concentration of 178 Z.C. Gazim et al. / Experimental Parasitology 129 (2011) 175–178

20%.The use of a pesticide composed of a mixture of a pyrethroid the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: (cypermethrin) and an organophosphate insecticide (chlorpyrifos) Ixodidae). Brazilian Archives of Biology and Technology 51, 1147–1152. Ferrarini, S.R., Duarte, M.O., Rosa, R.G., Rolim, V., Eifler-Lima, V.L., Von Poser, G., has contributed greatly to agricultural development, but poses Ribeiro, V.L.S., 2008. Acaricidal activity of limonene, limonene oxide and b- risks to the ecosystem (Zhou et al., 2011). In contrast, Chung- amino alcohol derivatives on Rhipicephalus (Boophilus) microplus. Veterinary samarnyart et al. (1991) pointed out that natural acaricides tend Parasitology 157, 149–153. Ferraz, A.B.F., Balbino, J.M., Zini, C.A., Ribeiro, V.L.S., Bordignon, S.A.L., von Poser, G., to have low toxicities to mammals, do not induce the rapid devel- 2010. Acaricidal activity and chemical composition of the essential oil from opment of resistance, and degrade rapidly in the environment. three Piper species. Parasitology Research 107, 243–248. In conclusion, this study shows that the essential oil of T. riparia Furlong, J., Martins, J.R., Prata, M.C.A., 2004. Controle estratégico do carrapato dos bovinos. A Hora Veterinária (Porto Alegre, RS) 23, 53–56. grown in southern Brazil showed high acaricidal activity against Gazim, Z.C., Amorim, A.C.L., Hovell, A.M.C., Rezende, C.M., Nascimento, I.A., Ferreira, the tick R. (B.) microplus. Our results showed that the essential oil G.A., Cortez, D.A.G., 2010. Seasonal variation, chemical composition, and of T. riparia is a promising biocontrol candidate for use against pes- analgesic and antimicrobial activities of the essential oil from leaves of Tetradenia riparia (Hochst.) Codd in Southern Brazil. Molecules 15, 5509–5524. ticide-resistant strains of R. (B.) microplus. However, complemen- Grisi, L., Massard, C.L., Moya Borja, G.E., Pereira, I.B., 2002. Impacto econômico das tary experiments to evaluate the acaricidal activity are needed, in principais ectoparasitoses em bovinos no Brasil. A Hora Veterinária 21, 8–10. particular in vivo tests using animal models. Martinez-Velazquez, M., Castillo-Herrera, G.A., Rosario-Cruz, R., Flores-Fernandez, J.M., Lopez-Ramirez, J., Hernandez-Gutierrez, R., Lugo-Cervantes, E. del C., 2010. Acaricidal effect and chemical composition of essential oils extracted from Acknowledgments Cuminum cyminum, Pimenta dioica and Ocimum basilicum against the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Parasitology Research (published online 24.09.2010). The authors thank the Coordenação de Aperfeiçoamento de Pes- Martins, R.M., 2006. 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