Acaricidal activity of the essential oil from Tetradenia riparia - Core

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Sep 24, 2010 - Water with 0.125% Colosso Pulverização® (cyper- methrin 0.25; chlorpyrifos ..... particular in vivo tests using animal models. Acknowledgments.
Experimental Parasitology 129 (2011) 175–178

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Acaricidal activity of the essential oil from Tetradenia riparia (Lamiaceae) 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 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 b

a r t i c l e

i n f o

Article history: Received 10 November 2010 Received in revised form 20 June 2011 Accepted 28 June 2011 Available online 5 July 2011 Keywords: Tetradenia riparia Acaricide Ixodidae Tick Rhipicephalus (Boophilus) microplus

a b s t r a c t Tetradenia riparia (Lamiaceae) is a well-known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of T. riparia essential oil (EO) against engorged females of Rhipicephalus (Boophilus) microplus (Acari; Ixodidae). For this purpose, nine serial concentrations (12.50%, 6.25%, 3.75%, 1.80%, 0.90%, 0.45%, 0.22%, 0.11%, and 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%, 0.114%, 0.057%, 0.028%, and 0.014% w/v). The results for AIT showed 100.00% and 2.05% mortality, 19.00 and 90.20% for the total number of eggs, egg-laying inhibition of 0.00% and 90.20%, hatchability inhibition of 0.00% and 70.23%, and product effectiveness of 100.00% and 2.89%, respectively. The AIT indicated that the LC50 and LC99.9, calculated using the Probit test, were for mortality (%) 0.534 g/mL (0.436–0.632) and 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 Brazil, the main tick species that undermines the productivity of cattle raising is Rhipicephalus (Boophilus) microplus. The damage caused by this tick to South American cattle exceeds two billion dollars a year (Grisi et al., 2002). Primarily by biting, it affects the production of meat and milk but the tick also injects toxins into the host and transmits infectious agents (Olivo et al., 2008). The control of cattle ticks is usually done using conventional chemicals including synthetic pyrethroids (SP), organophosphates (OP), and amitraz (Am). These cause the rapid development of resistance to the active principle. Further, their use has caused great concern

⇑ Corresponding author. E-mail address: [email protected] (D.A.G. Cortez).

in society and government, by harming the animals themselves and humans who consume the products from these animals (Chagas et al., 2003). Each time that ticks survive an application of insecticide, they transmit genetic information to later generations about how to survive that product (Furlong et al., 2004). The use of plant extracts in tick control has also been the focus of extensive research (Martinez-Velazquez et al., 2010). Tetradenia riparia (Hochstetter) Codd, a member of the family Lamiaceae, is a shrub common throughout Africa. In South Africa, it is one of the most popular herbs and medicinal plants (Van Puyvelde and De Kimpe, 1998). For decades, T. riparia has been the subject of 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 et al., 1992); insecticide (Weaver et al., 1994); antimalarial (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

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(Omolo et al., 2004). To date, no studies reported in the literature on the acaricidal activity of this plant. Thus the present experiment was to evaluate the acaricidal activity of the essential oil from fresh leaves of T. riparia against the acari of R. (B.) microplus. 2. Materials and methods 2.1. Plant material Plant material (leaves) of T. riparia was collected in a medicinalplant garden at the Universidade Paranaense (Unipar), northwestern Paraná State, Brazil. A voucher specimen was authenticated and deposited at the herbarium of the University Educational Paranaense (HEUP), under number 2502. To obtain the essential oil of T. riparia, fresh leaves were collected between 06:30 and 08:00 a.m. during summer, between 21/12/2008 and 21/03/2009, and hydrodistilled for 3 h using a modified Clevenger-type apparatus. The distilled oils were collected and dried over anhydrous sodium sulfate and stored in a freezer (Gazim et al., 2010). 2.2. Ticks About 300 engorged females of R. (B.) microplus were collected from naturally infested cattle on farms in Tapejara city, Paraná, which had suspended the use of acaricidal treatment for at least 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. 2.3. Bioassays (AIT and LPT) For the acaricidal test (AIT), we made serial dilutions (12.50%, 6.25%, 3.75%, 1.80%, 0.90%, 0.45%, 0.22%, 0.11%, and 0.055% w/v) of essential oil of T. riparia. For the larval packet test (LPT) the concentrations used were 100.00%, 25.00%, 12.50%, 6.25%, 3.65%, 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), described by Farias et al. (2007). The AIT was described by Drummond et al. (1976), where groups of 30 engorged females were weighed and immersed for 5 min in the respective dilutions (10 mL) in a 50-mL beaker which was gently agitated (three times) at room temperature. The emulsifying solution (2% Tween) was used as the negative control (Farias et al., 2007; Rosado-Aguilar et al., 2010). Water with 0.125% Colosso PulverizaçãoÒ (cypermethrin 0.25; chlorpyrifos 0.25 and citronellal 0.01 g/mL) was used as a positive control. Ticks were removed from the solutions, dried, and each group placed in a separate Petri dish. The Petri dishes were incubated at 27–28 °C and 70–80% relative humidity. After 14 days, the number of females laying eggs was recorded, and the eggs were collected, weighed, and observed. The eggs were placed in glass tubes, incubated at 27–28 °C and 70–80% relative humidity, and after 21 days the percentage of hatched eggs was calculated. The effectiveness of the product (PE) was calculated using the mathematical formulas below, described by Drummond et al. (1973). Each treatment contained three replicates:

Reproduction estimated ðREÞ ¼ ⁄

egg weight  % hatchability  20:000 weight of females

Constant indicating the number of eggs present in 1 g of egg laying.

Effectiveness of the product ðPEÞ ¼

RE ðcontrol groupÞ  RE ðtreated groupÞ  100 RE ðcontrol groupÞ

To implement the LPT test, larvae were obtained from engorged females of R. (B.) microplus, and allowed to rest unfed for 14–21 days following hatchability prior to their use. The larvae were exposed to test solutions in filter-paper envelopes (2  2 cm), containing micropores to allow better ventilation (Fernandes et al., 2008). The following treatments were used: (1) envelopes of dry filter paper; (2) envelopes of filter paper moistened with a negative control (solution of 2% Tween 80 in distilled water); (3) positive controls consisting of water with 0.125% Colosso PulverizaçãoÒ (cypermethrin 0.25; chlorpyrifos 0.25 and citronellal 0.01 g/mL) and (4) envelopes moistened with 2 mL of each concentration of essential oil of T. riparia to be tested. After each envelope was treated and larval ticks inserted, the opening was folded over (10 mm) and re-sealed with a metallic clip, with its identification mark (solution and concentration tested) on the outside. The packets were placed in the BOD incubator at a temperature of 27–28 °C and 85–95% relative humidity for 24 h. The envelopes were then opened and inspected, using a stereomicroscope, to record the number of live and dead larvae, and any toxicological effects observed. Each treatment contained three replicates:

Corrected percent mortality ¼

% test mortality  % control mortality  100 100  % control mortality

2.4. Statistical analyses The experimental design was completely randomized. The data were processed and subjected to analysis of variance (ANOVA), and differences between means were determined by Tukey test at 5% significance level. Lethal concentrations (LC) to kill 50% and 99% of larvae and their respective 95% confidence intervals (CI) were calculated by probit analysis (software R version 11.2). 3. Results The essential oil of T. riparia was analyzed by Gazim et al. (2010), and 31 compounds, comprising 97.17% of the oil, were identified. The predominant class in this oil was oxygenated sesquiterpenes: 14-hydroxy-9-epi-cariophyllene (18.03%), cismuurolol-5-en-4-a-ol (11.73%), ledol (7.18%), and a-cadinol (4.90%), followed by monoterpene hydrocarbons: limonene (3.69%), and oxygenated: fenchone (12.87%). The percentages obtained for in vitro efficacy of the oil of T. riparia against R. (B.) microplus are shown in Table 1. In the AIT, the efficacy of treatment against engorged females was assessed by measuring mortality of gravid females, total number of eggs, egg weight, percentage of hatchability, and product efficiency (Table 1). Table 2 shows the percent mortality of R. (B.) microplus larvae exposed to different concentrations of T. riparia essential oil through the larval packet test (LPT). In dilutions of 100%, 50%, and 25% the oil killed 100% of the larvae, indicating a maximum 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 no significant differences between them. However, all these results differed significantly from the negative control (8.00%). The LC50 and LC99.9 (Table 3) calculated using the Probit test, were for% mortality 0.534 g/mL (0.436–0.632) and 1.552 g/mL (1.183–1.92), for total number of eggs 0.449 g/mL (0.339–0.558) and 1.76 g/mL (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 mortality (LPT) was 1.222 g/mL (0.655–1.788) and 11.382 g/mL (7.84–14.91), respectively.

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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 12.5 6.25 3.75 1.80 0.90 0.45 0.22 0.11 0.056 C2

100 ± 0.00 100 ± 0.00 100 ± 0.00 100 ± 0.00 100 ± 0.00 76.68 ± 15.27a 53.33 ± 15.27 b 23.33 ± 5.77c 10.00 ± 10.00d 2.05 ± 2.30e 1.10 ± 1.90d

0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 19.00 ± 2.22a 35.87 ± 3.00b 60.27 ± 2.93c 80.12 ± 7.30d 90.20 ± 9.42e 91.30 ± 5.08e

0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.068 ± 0.010a 0.092 ± 0.010a 0.066 ± 0.010a 0.075 ± 0.010a 0.095 ± 0.010a 0.075 ± 0.010a

0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 34.87 ± 6.23a 24.13 ± 1.68a 45.47 ± 2.08a 59.47 ± 2.79b 70.23 ± 2.25c 73.87 ± 2.24c

100 ± 0.00 100 ± 0.00 100 ± 0.00 100 ± 0.00 100 ± 0.00 64.27 ± 10.66a 68.28 ± 5.63a 45.63 ± 7.25b 25.72 ± 10.01c 2.89 ± 3.01d 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 larvae exposed to different concentrations of T. riparia essential oil. T. riparia essential oil (% w/v)

% Average mortality

C1 100.00 50.00 25.00 12.50 6.25 3.65 1.82 0.91 0.45 0.228 0.114 0.057 0.028 0.014 C2

99.6 ± 1.22a 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 97.60 ± 1.21a 96.30 ± 1.20a 94.60 ± 1.20a 74.60 ± 1.20b 62.30 ± 1.19b 58.60 ± 1.17b 56.60 ± 1.16b 46.60 ± 1.16c 44.60 ± 1.16c 24.60 ± 1.16d 10.60 ± 1.16e 8.00 ± 0.76e

C1: positive control (eater 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 3 Lethal concentration LC99.9 and LC50 of T. riparia essential oil for R. (B.) microplus and respective confidence intervals, calculated starting from the interpolation of the acaricidal test (AIT) mortality of gravid females (%), total number of eggs and hatchability results, and larval packet test (LPT) by Probit analysis.

Acaricidal test (AIT) Mortality of gravid females (%) Total number of eggs Hatchability (%) Larval packet test (LPT) Mortality larvae (%)

LC50 (g/mL) (CI)

LC99.9 (g/mL) (CI)

0.534 (0.436–0.632)

1.552 (1.183–1.92)

0.449 (0.339–0.558) 0.114 (0.0–0.31)

1.76 (1.27–2.248) 2.462 (1.501–3.422)

1.222 (0.655–1.788)

11.382 (7.84–14.91)

LC50: lethal concentration 50%; LC99.9: lethal concentration 99.9%; CI: confidence intervals.

4. Discussion The analyses of the essential oil from T. riparia by GC–MS identified a mixture of sesquiterpenes (50.30%) and monoterpenes (19.52%). The essential oil showed intense activity against different

stages of R. (B.) microplus, and the mortality rate of the ticks was dose-dependent. Many essential oils and their components, mainly monoterpenoids and sesquiterpenoids, show repellent, chemosterilant, antifeeding, and biocidal activities against the cattle tick R. (B.) microplus (Facey et al., 2005). However, the chemical constituents of essential oils from other plant species differ from those found in the essential oil of T. riparia. For example, the oil of T. riparia killed 100% of gravid female ticks at concentrations from 1.8% to 12.5%. Similarly, the studies of Martins (2006) with essential oil of Cymbopogon winterianus (citronella or Java grass), which is rich in monoterpenes (41.15% citronellal), showed 100% mortality at concentrations from 12.5% to 50.0%. Chagas et al. (2002) investigated the action of monoterpene-rich essential oil from three species of Eucalyptus: Eucalyptus citriodora (94.9% citronellal), Eucalyptus globulus (9.93% a-pinene), and Eucalyptus staigeriana (24.78% DL-limonene), in five different concentrations, against gravid female ticks. The results showed that E. citriodora, E. globulus and E. staigeriana killed 100% of females at mean concentrations of 17.50%, 15% and 12.5%, respectively. Regarding the inhibitory action of T. riparia essential oil on oviposition, there was a 100% reduction of egg laying with oil concentrations from 1.80% to 12.5%. This effect was also evaluated by Olivo et al. (2008), who investigated the effect of the essential oil from C. nardus containing high levels of monoterpenes (50.0% citronellal), and observed 90% inhibition of oviposition at concentrations of 100%, 50%, 25% and 10%. Also, Ribeiro et al. (2010) found that essential oil from H. ringens, which has a high content of oxygenated monoterpenes (86.0% Pulegone), inhibited egg laying by 76.4% and 48.2% at concentrations of 5.0% and 2.5%, respectively. Another parameter evaluated was the influence of T. riparia essential oil on the percentage of hatching of larvae. The results showed 100% inhibition of hatching at concentrations ranging from 1.8% to 12.5%. Ferrarini et al. (2008) evaluated the effect of limonene (monoterpene hydrocarbons) in inhibiting egg hatching of R. (B.) microplus, at concentrations from 0.25% to 1.0%, and obtained 91% to 100% inhibition. Taken together, the results showed that oil has a significant potential to control the larvae of R. (B.) microplus. Ferraz et al. (2010) observed that for the essential oil of Piper amalago, which contained high levels of monoterpenes (20.52% limonene) and sesquiterpenes (11.18% zingiberene), the most 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% cyclocolorenone), on the larvae of R. (B.) microplus. One hundred percent of the larvae were killed at concentrations of 2.5%, 1.25% and 0.625%. Chagas et al. (2002) also observed that the essential oil of E. staigeriana killed 100% of the larvae at a concentration of 10%, and E. globulus had the same effect at a concentration of

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20%.The use of a pesticide composed of a mixture of a pyrethroid (cypermethrin) and an organophosphate insecticide (chlorpyrifos) has contributed greatly to agricultural development, but poses risks to the ecosystem (Zhou et al., 2011). In contrast, Chungsamarnyart et al. (1991) pointed out that natural acaricides tend to have low toxicities to mammals, do not induce the rapid development of resistance, and degrade rapidly in the environment. In conclusion, this study shows that the essential oil of T. riparia grown in southern Brazil showed high acaricidal activity against the tick R. (B.) microplus. Our results showed that the essential oil of T. riparia is a promising biocontrol candidate for use against pesticide-resistant strains of R. (B.) microplus. However, complementary experiments to evaluate the acaricidal activity are needed, in particular in vivo tests using animal models. Acknowledgments The authors thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Ensino Superior (CAPES) and the Conselho Nacional de Pesquisas e Desenvolvimento (CNPq) for the financial support. References Campbell, W.E., Gammon, D.W., Smith, P., Abrahams, M., Purves, T., 1997. Composition and antimalarial activity in vitro of the essential oil of Tetradenia riparia. Planta Medica 63, 270–272. Chagas, A.C.S., Passos, W.M., Prates, H.T., Leite, R.C., Furlong, J., Fortes, I.C.P., 2002. Acaricide effect of Eucalyptus spp. essential oils and concentrated emulsion on Boophilus microplus. Brazilian Journal of Veterinary Research and Animal Science 39, 247–253. Chagas, A.C.S., Leite, R.C., Furlong, J., Prates, H.T., Passos, W.M., 2003. Sensibilidade do carrapato Boophilus microplus a solventes. Ciência Rural 33, 109–114. Chungsamarnyart, N., Jiwajinda, S., Ratanakreetakul, C., 1991. Practical extraction of sugar apple seeds against tropical cattle ticks. Kasetsart Journal: Natural Science 25, 101–105. Drummond, R.O., Ernst, S.E., Trevino, J.L., Gladney, W.J., Graham, O.H., 1973. Boophilus annulatus and Boophilus microplus: laboratory tests for insecticides. Journal of Economic Entomology 66, 130–133. Drummond, R.O., Ernst, S.E., Trevino, J.L., Gladney, W.J., Graham, O.H., 1976. Tests of acaricides for control of Boophilus annulatus and B. microplus. Journal of Economic Entomology 69, 37–40. Facey, P.C., Porter, R.B.R., Reese, P.B., Williams, L.A.D., 2005. Biological activity and chemical composition of the essential oil from Jamaican Hyptis verticillata Jacq.. Journal of Agriculture and Food Chemistry 53, 4774–4777. Farias, M.P.O., Sousa, D.P., Arruda, A.C., Arruda, M.S.P., Wanderley, A.G., Alves, L.C., Faustino, M.A.G., 2007. Eficácia in vitro do óleo da Carapa guianensis Aubl. (andiroba) no controle de Boophilus microplus (Acari: Ixodidae). Revista Brasileira de Plantas Medicas Botucatu 9, 68–71. Fernandes, F.F., Bessa, P.A.D., Freitas, E.P.S., 2008. Evaluation of activity of the crude ethanolic extract of Magonia pubescens St. Hil (Sapindaceae) against larvae of

the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: 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., Ribeiro, V.L.S., 2008. Acaricidal activity of limonene, limonene oxide and bamino alcohol derivatives on Rhipicephalus (Boophilus) microplus. Veterinary 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., 2010. Acaricidal activity and chemical composition of the essential oil from three Piper species. Parasitology Research 107, 243–248. 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. Gazim, Z.C., Amorim, A.C.L., Hovell, A.M.C., Rezende, C.M., Nascimento, I.A., Ferreira, G.A., Cortez, D.A.G., 2010. Seasonal variation, chemical composition, and analgesic and antimicrobial activities of the essential oil from leaves of Tetradenia riparia (Hochst.) Codd in Southern Brazil. Molecules 15, 5509–5524. Grisi, L., Massard, C.L., Moya Borja, G.E., Pereira, I.B., 2002. Impacto econômico das principais ectoparasitoses em bovinos no Brasil. A Hora Veterinária 21, 8–10. 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 Cuminum cyminum, Pimenta dioica and Ocimum basilicum against the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Parasitology Research (published online 24.09.2010). Martins, R.M., 2006. Estudo in vitro da ação acaricida do óleo essencial da gramínea Citronela de Java (Cymbopogon winterianus Jowitt) no carrapato Boophilus microplus. Revista Brasileira de Plantas Medicinais 8, 71–78. Olivo, C.J., Carvalho, N.M., Silva, J.H.S., Vogel, F.F., Massariol, P., Meinerz, G., Agnolin, C., Morel, A.F., Viau, L.V., 2008. Óleo de citronela no controle do carrapato de bovinos. Ciência Rural 38, 406–410. Omolo, M.O., Okinyo, D., Ndiege, I.O., Lwande, W., Hassanali, A., 2004. Repellency of essential oils of some Kenyan plants against Anopheles gambiae. Phytochemistry 65, 2797–2802. Ribeiro, V.L.S., Avancini, C., Gonçalves, K., Toigo, E., Von Poser, G., 2008. Acaricidal activity of Calea serrata (Asteraceae) on Boophilus microplus and Rhipicephalus sanguineus. Veterinary Parasitology 151, 351–354. Ribeiro, V.L.S., Santos, J.C., Bordignon, S.A.L., Apel, M.A., Henriques, A.T., von Poser, G.L., 2010. Acaricidal properties of the essential oil from Hesperozygis ringens (Lamiaceae) on the cattle tick Riphicephalus (Boophilus) microplus. Bioresource Technology 101, 2506–2509. Rosado-Aguilar, J.A., Aguilar-Caballero, A., Rodriguez-Vivas, R.I., Borges-Argaez, R., Garcia-Vazquez, Z., Mendez-Gonzalez, M., 2010. Acaricidal activity of extracts from Petiveria alliacea (Phytolaccaceae) against the cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Veterinary Parasitology 168, 299– 303. Van Puyvelde, L., De Kimpe, N., 1998. Tetradenolide, an a-pyrone from Tetradenia riparia. Phytochemistry 49, 1157–1158. Weaver, D.K., Dunkel, F.V., Cusker, J.L., Van Puyvelde, L., 1992. Oviposition patterns in two species of bruchids (coleopteran: bruchidae) as influenced by the dried leaves of Tetradenia riparia, a perennial mint (lamiales: lamiaceae) that suppresses population size. Environmental Entomology 21, 1123–1129. Weaver, D.K., Dunkel, F.V., Van Puyvelde, L., Richards, D.C., Frizgerald, G.W., 1994. Toxicity and protectant potential of the essential oil of Tetradenia riparia (lamiales, lamiaceae) against zabrotes-subfasciatus (col, bruchidae) infesting dried pinto beans (fabales, leguminosae). Journal of Applied Entomology 118, 179–196. Zhou, S., Duan, C., Michelle, W.H.G., Yang, F., Wang, X., 2011. Individual and combined toxic effects of cypermethrin and chlorpyrifos on earthworm. Journal of Environmental Sciences 23, 676–680.