Toxicity of Piper aduncum L. (Piperales: Piperaceae) from the Amazon forest for the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Veterinary Parasitology 164 (2009) 267–274

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Toxicity of Piper aduncum L. (Piperales: Piperaceae) from the Amazon forest for the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) Wilson Castro Silva a,*, Joa˜o Ricardo de Souza Martins b, Hellen Emı´lia Menezes de Souza c, Horacio Heinzen d, Maria Veroˆnica Cesio d, Mauricio Mato d, Francine Albrecht a, Joa˜o Lu´cio de Azevedo a, Neiva Monteiro de Barros a a

Laborato´rio de Controle de Pragas, Instituto de Biotecnologia, Universidade de Caxias do Sul. Rua Francisco Getu´lio Vargas, 1130, Petro´polis, Caxias do Sul - RS 95070-560, Brazil b Laborato´rio de Parasitologia, Instituto de Pesquisas Veterina´rias Deside´rio Finamor, Estrada do Conde, 6000, Eldorado do Sul - RS 92990-000, Brazil c Programa Amazonas de Integrac¸a˜o da Cieˆncia no Interior (PAICI/FAPEAM), Universidade do Estado do Amazonas/Centro Universita´rio Nilton Lins. Av. Carvalho Leal, 1777, Cachoeirinha, Manaus - AM 69000 000, Brazil d University of the Republic, Faculty of Chemistry, Box 1157, Montevideo 11800, Uruguay

A R T I C L E I N F O

A B S T R A C T

Article history: Received 4 December 2008 Received in revised form 18 March 2009 Accepted 8 June 2009

The mortality of 14–21-day-old Rhipicephalus (Boophilus) microplus larvae, and the mortality and fertility of groups of engorged adult females exposed to different concentrations of hexane, ethyl acetate and ethanol extracts of spiked pepper (Piper aduncum) were evaluated, using a completely randomized design with five treatment groups, two control groups, and two replicates for the larvae and five replicates for the adult females. Similar methodology was used to investigate the toxicity of the essential oil hydro-distillate (94.84% dillapiole) obtained from the P. aduncum crude hexane extract. The LC50 of the hexane extract was 9.30 mg ml1 for larvae and the reproduction reduction ranged from 12.48% to 54.22%, while 0.1 mg/ml1 of the essential oil induced 100% mortality in larvae. Literature reports on natural products active against R. microplus were listed and compared with the results presented here. These results indicate that P. aduncum extracts, and particularly its essential oil, are potential alternative control agents for R. microplus. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Cattle ticks Rhipicephalus (Boophilus) microplus Piper aduncum Plants extracts

1. Introduction Brazil has the second largest herd of beef cattle in the world, with 205.9 million head in 2006, second only to India, which does not market them commercially for religious reasons (IBGE, 2008). However, the growth potential of this important economic activity is threatened by several factors harmful to livestock, like the tick Rhipicephalus (Boophilus) microplus.

* Corresponding author. Tel.: +55 54 3218 2149. E-mail addresses: [email protected], [email protected] (W.C. Silva). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.06.006

The cattle tick R. microplus L. (Acari: Ixodidae) originated in Asia but is now widely distributed worldwide, infecting cattle (Johnston et al., 1986). This tick causes large losses to ranchers because as well as being a disease vector, it reduces reproductive efficiency, meat and milk production and can produce ‘pinholes’ which reduce the hide value (Cordove´s, 1997). In Brazil, the annual economic losses caused by R. microplus have been estimated to be about one billion US dollars during the 1980s (Horn, 1987) and have now risen to about two billion US dollars. Cattle ticks may be controlled using synthetic acaricides (Klafke et al., 2006; Li et al., 2007; Barre´ et al., 2008; Davey et al., 2008; Miller et al., in press) but these can have

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undesirable effects on other organisms and the environment. Ultimately they become ineffective as ticks quickly develop resistance to these chemicals (Chagas et al., 2002b; Chagas, 2004; George et al., in press). Such considerations have led to the search for alternative control methods, which have included biocontrol by nematodes (Vasconcelos et al., 2004) and entomopathogenic fungi such as Beauveria and Metarhizium (Mwangi et al., 1995; Zhioua et al., 1997; Bittencourt et al., 1999; Kaaya and Hassan, 2000; Gindin et al., 2001; Fernandes et al., 2002; Polar et al., 2005; Bahiense et al., 2006; Leemon et al., 2008). Ticks can also be controlled using plant extracts, which are useful alternatives, as natural products degrade fast in the environment and resistance is developed slower than against synthetic acaricides (Herna´ndez et al., 1987; Gupta et al., 2000; Choudhury, 2001; Ismail et al., 2002; Borges et al., 2003; Pereira and Famadas, 2006; Silveira Novelino et al., 2007; Fernandes and Freitas, 2007; Ribeiro et al., 2008b; Srivastava et al., in press). The huge plant diversity of the Amazonian forest enables research into new products for tick control. Ethnobotanical surveys in the Manaos region for insecticidal uses of plants and their extracts, indicated that spiked pepper (Piper aduncum L. Piperales: Piperaceae), a widely distributed plant in Central America, Southeast Asia and North and Northeastern Brazil and in the Amazonian forest, has been used as an insecticide by the population (Maia et al., 2001). The leaves and stems of P. aduncum contain an essential oil composed mainly of dillapiole (5-allyl 6,7-dimethoxy 1,3benzodioxole) (Pino et al., 2004; Walia et al., 2004) which has been demonstrated to have synergistic effects with several natural pesticides, and also to have insecticidal, bactericidal and fungicidal activity (Maia et al., 1998; Silva et al., 2007; Rafael et al., 2008). This work reports the toxicity of P. aduncum extracts for different development stages of R. microplus, and the potential of the extracts to control the tick is evaluated. The antiacarid effect of the oil and extracts of P. aduncum is compared to toxicity levels of other chemical and natural products described in the literature. 2. Materials and methods 2.1. General Solvents were reagent grade and distilled from glass prior to use. Precoated Thin Layer Chromatography (TLC) plates were from Machey Nagel. For hydrodistillation, a Clevenger apparatus was used (World Health Organization, 1998). Gas Chromatography/Flame Ionization Detector (GC/FID): A GC-Shimadzu 17-A equipped with a capillary column SE-52 (60 8C 8 min–3 8C/min–180 8C– 20 8C/min–250 8C–25 min at 250 8C), using He as carrier and FID detection (320 8C) was employed. The same temperature program was used in the Gas Chromatography/Mass Spectrometry (GC/MS) determination. GC/MS was run on a Shimadzu GC-17 gas chromatograph with a SE 52 capillary column (25 m, 0.25 mm i.d., 0.25 mm thickness), using He as carrier, coupled with an Electronimpact Ionization (EI) quadrupole MS Shimadzu QP5050, detector. Adams’ library of mass spectra was employed to identify the essential oil components.

2.2. Collection and processing of P. aduncum leaves Leaves from P. aduncum were collected at km 27 of the AM-010 road in the Ducke Reserve in Manaus in the Brazilian state of Amazonas (s028 54.778; w598 58.802). The leaves were dried in a greenhouse at 37 8C for five days and then ground and packed at 20–25 8C (Prista et al., 1981). 2.3. Extraction and analysis of P. aduncum dry leaves The powdered leaf was Soxhlet extracted using a sequence of solvents of increasing polarity (hexane, ethyl acetate and ethanol) for 24 h each. After extraction the solutions were decanted, filtered and the solvents removed by evaporation under reduced pressure. The resulting crude extracts were stored at 4 8C. A volatile fraction was obtained from 3.68 g of the crude hexane extract by hydro-distillation in a Clevenger device. The hydro-distillate was analyzed by GC/FID and GC/MS. 2.4. Tick collection R. microplus was collected from January to March of 2007 from cattle belonging to a herd located at the Deside´rio Finamor Institute of Veterinary Research in Eldorado do Sul-RS-Brazil. Female ticks, in the final stage of engorgement were collected from the ground, after falling from the cattle, and used in the biological tests within 24 h after the collection. 2.5. Bioassay Three hundred fifty (350) engorged females were separated into groups of ten (10), weighed, in order to get a sample of uniform weight for immersion in each solution for five minutes. The testing solutions were the three extracts (hexane, ethyl acetate, and ethanol), which were solubilized in Tween-80 5% (v/v) at concentrations of 5 mg ml1, 25 mg ml1, 50 mg ml1, 75 mg ml1, and 100 mg ml1. Another two groups were employed for control purposes: One was immersed in Tween-80 5% (v/ v), and the other in distilled water (Drummond et al., 1973). After treatment, each group was placed in a Petri plate and maintained at 27  1 8C and 80  5% relative humidity. The mortality rates were evaluated daily over a sixday period. Oviposition rates were evaluated over a 15-day period. Reproductive efficiency and reproduction control were evaluated over 15 days, after weighing the eggs, using the following formula (Stendel, 1980). Inhibition of oviposition (IO) EW IO ¼ IFW %IO ¼

IO ðcontrolÞ  IO ðtreatedÞ  100 IO ðcontrolÞ

Reproductive efficiency (RE) EW  %E %RE ¼ IFW

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2.6. Statistical analysis

Control of reproduction (CR) REC  RET  100 %CR ¼ REC where (%IO) = percentage inhibition of oviposition; (%RE) = percentage reproductive efficiency, IFW = initial female weight, EW = egg weight, (%E) = percentage eclosion, (%CR) = percentage control of reproduction, REC and RET = reproductive efficiency of controls and treated group respectively. The toxicity of hexane, ethyl acetate and ethanol extracts of P. aduncum against larvae was tested at concentrations of 1 mg ml1, 5 mg ml1, 10 mg ml1, 15 mg ml1 and 20 mg ml1 using a modification of a previously described methodology (Shaw, 1966; Souza et al., 2006). 100 Larvae around 14 and 21 days old, obtained by oviposition of untreated engorged female ticks, were put into a previously prepared 5 ml syringe. The syringe was cut next to the needle’s region leaving an orifice of approximately 0.1 mm diameter in the middle. Before the immersion of the larvae, the orifice was closed with a fine weft fabric fixed with an orthodontic rubber band. The larvae were immersed in the extracts for five minutes and maintained at 27  1 8C and 80  5% relative humidity. This procedure was repeated for each concentration. The same basic procedure was used for two controls using 5% (v/v) aqueous Tween80 and distilled water. After 24 h the number of live and dead larvae was counted and the percentage mortality was calculated as %mortality = number of dead larvae/total number of larvae  100 (FAO Plant Protection Bulletin, 1971). The same methodology was used to evaluate the toxicity of the essential oil, diluted in 95% ethanol, at concentrations of 0.1 mg ml1, 0.5 mg/ml1 and 1 mg/ ml1 on larvae (Chagas et al., 2003).

The experiments were totally randomized with five treatments, five replicates and two control groups for the engorged females and five treatments, two replicates and two control groups for the larvae. Since the two control groups were not statistically different, Tween-80 control was used in the analysis. The lethal concentration 50% (LC50) and the 95% confidence intervals (CI95%) were calculated according to the Probit analysis (Finney, 1971) using the Toxrat1 program (Toxrat1, 2003), which was also used for constructing the dose–response curves. In order to evaluate only the mortality due to the extracts in the CL50 calculation, corrections for the contribution for natural mortality were performed using Abbott’s formula (Abbott, 1925). Analysis of variance (ANOVA) of the data (Zar, 1984) was carried out using the Statistical Package for the Social Sciences (SPSS) for Windows, version 12.0. 3. Results Table 1 shows the initial female weight (IFW), egg weight (EW), percentage reproductive efficiency (%RE) and percentage control of reproduction (%CR) for engorged female ticks exposed to different concentrations of P. aduncum leaf extracts produced with three different organic solvents. All the three extracts showed a direct concentration-dependent effect on the %CR. At the higher concentration (100 mg ml1) the %CR was 61% for the ethyl acetate extract and 54% for the hexane extract and the ethanol extract was the less effective. Nevertheless the results (Table 1) show that there were not statistical differences between all three extracts at 50–100 mg ml1 (Tukey test, P > 0.05). The ethyl acetate (CR50 = 61.41 mg ml1) and hexane (CR50 = 74.54 mg ml1) extracts were not statistically different in relation at the mean control of reproduction

Table 1 Initial female weight (IFW), egg weight (EW), percentage reproductive efficiency (%RE) and percentage control of reproduction (%CR) for engorged female Rhipicephalus microplus ticks exposed to different concentrations of Piper aduncum. Extract (mg ml1)

IFW (g)

EW (g)

%RE

Hexane 5 25 50 75 100

3.24  0.17 3.46  0.05 3.29  0.13 3.21  0.16 3.26  0.12

1.41  0.21 1.26  0.33 1.10  0.10 0.99  0.29 0.94  0.20

41.02  5.69 33.76  9.53 26.30  4.26 22.54  4.38 21.52  4.96

af baf cb cb c

12.48  12.53 a 28.70  17.53 ba 43.96  8.84 cb 52.12  7.92 c 54.22  9.85 c

Ethyl acetate 5 25 50 75 100

1.96  0.12 1.86  0.18 1.89  0.19 1.98  0.20 1.87  0.12

0.78  0.06 0.62  0.15 0.53  0.13 0.51  0.13 0.41  0.09

38.60  3.51 29.33  5.98 23.27  4.71 20.09  5.65 16.66  3.92

af ba cb cb c

11.52  12.68 32.77  17.55 46.66  11.01 53.95  15.04 61.81  11.53

Ethanol 5 25 50 75 100

3.35  0.26 3.29  0.19 3.38  0.13 3.26  0.12 3.13  0.09

1.44  0.18 1.35  0.08 1.24  0.07 1.15  0.09 0.99  0.11

40.44  5.80 38.72  2.73 32.96  2.20 32.02  2.41 28.84  3.01

af baf cab cb c

13.14  14.08 a 17.04  7.49 ba 29.50  4.00 cab 31.40  6.39 cb 38.08  8.71 c

Control

2.20  0.14

0.96  0.10

43.80  5.24 f

Means followed by the same letter were not significantly different by the Tukey test at P = 0.05.

%CR

–

a ba cb cb c

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Table 2 Percentage mortality (%M) and percentage inhibition of oviposition (%IO) for engorged female Rhipicephalus microplus ticks exposed to different concentrations of Piper aduncum leaf extracts. Extract

Concentration (mg ml1)

Control

5

Hexane Ethyl acetate Ethanol

25

50

75

100

%M

%IO

%M

%IO

%M

%IO

%M

%IO

%M

%IO

%M

%IO

4 4 4

0 0 0

6 6 4

10.14 7.01 10.56

10 12 6

25.11 23.04 14.78

12 20 6

31.85 34.80 23.84

16 22 10

37.05 40.68 26.66

16 a 22 a 10 a

39.57 a 46.78 a 35.02 a

Means followed by the same letter were not significantly different by the Tukey test at P = 0.05.

Table 3 Percentage mortality (%M) for Rhipicephalus microplus larvae exposed to different concentrations of Piper aduncum leaf extracts. Extract

Control

Concentration (mg ml1) 1

5

10

15

20

Hexane Ethyl acetate Ethanol

1.65 0 0

11.40 2.00 26.32

18.26 7.26 31.05

55.19 13.31 33.05

68.36 14.46 37.11

70.42 a 17.25 b 40.47 c

Means followed by the same letter were not significantly different by the Tukey test at P = 0.05.

concentration (CR50). Both control test yielded the same %CR and %RE, and therefore, only the results from the Tween-80 control are reported. Table 2 shows the mortality percentage (%M) and percentage inhibition of oviposition (%IO) for engorged female ticks exposed to the extracts. However, at the concentrations assayed, the three extracts were not toxic enough to determine LC50 values for the engorged female ticks. On the other hand, the toxicity to larvae was statistically different for the three extracts. Particularly the ethanol extract was significantly more toxic to the larvae than the ethyl acetate one, but the more active it was the hexane extract as it is show in Table 3. The mean LC50 value of this extract was 9.30 mg ml1 (Fig. 1). The hexane extract was submitted to hydro-distillation, yielding 6.8% of a volatile oil. The composition of the essential oil, determined by GC–MS and Kovats Indexes

Fig. 1. Determination of Piper aduncum hexane extract concentration 50% (LC50) for Rhipicephalus microplus larvae.

lethal

(Fig. 2), proved to be almost pure dillapiole (94.84%), plus small amounts of sesquiterpenes: neorodiol (0.74%), globulol (0.65%), spathulenol (0.64%), and other shiquimates: croweacin (1.91%), apiole (0.38%). The essential oil produced 100% larval mortality at all the concentrations tested.

Fig. 2. GC–MS Chromatogram showing the composition of the P. aduncum essential oil. (1) Nerodiol (0.74%); (2) globulol (0.65%); (3) spathulenol (0.64%); (4) croweacin (1.91%); (5) dillapiole (94.84%); (6) apiole (0.38%).

W.C. Silva et al. / Veterinary Parasitology 164 (2009) 267–274

4. Discussion The toxicity towards R. microplus larvae showed by the P. aduncum hexane extract is mainly due to the essential oil which contains large amounts of dillapiole, a phenylpropanoid with reported insecticidal properties (Maia et al., 1998; Bhuiyan et al., 2001; Fazolin et al., 2005). The composition of the volatile fraction of the hexane extract in the present study is similar to that reported by Maia et al. (2001), who extracted essential oil from the leaves of samples of P. aduncum collected in different parts of the Brazilian Amazon. Dillapiole has been described as the major constituent compound of P. aduncum essential oil (Gottlieb et al., 1981; Pino et al., 2004; Oliveira et al., 2005; Rali et al., 2007), although there have been reports where the sesquiterpene E-nerolidol (peruviol) is the major compound (Mesquita et al., 2005; Oliveira et al., 2006). The constituents of volatile oils can vary according to the environmental conditions, such as climate and soil type and management. According to our results, in the essential oil, dillapiole is the major component but there are minor amounts of terpenoids (isoprenoids) that are also volatile secondary metabolites with known insecticidal activity (Viegas Ju´nior, 2003; Omar et al., 2007; Geris et al., 2008) which alter development and metabolism in several insect species and produce physiological disturbances that may lead to inhibition of reproduction or death due to interference with feeding and growth (Schmutterer, 1990). The combination of chemicals with different types of insecticide action usually lowers the LD50 of the whole product and hampers the development of resistance of the acarid. The essential oil obtained from the hexane extract was toxic to R. microplus larvae at 0.1 mg ml1. This concentration induced 100% mortality of R. microplus larvae, which (Table 4) was the same mortality induced by the essential oil from Eucalyptus staigeriana at a concentration of 10% (Chagas et al., 2002a) and Drimys brasiliensis at a concentration of 0.025% (Ribeiro et al., 2008b); Cymbopogon winterianus at a concentration of 4.1% induced 50% mortality (Martins, 2006). The insecticidal activity of the essential oil from P. aduncum has been reported previously. It induced 100% mortality in yellow mealworm (Tenebrio molitor) larvae (Fazolin et al., 2007) and in adult cowpea weevils (Callosobruchus maculatus) (Pereira, 2006). Chagas et al. (2003) reported that engorged female R. microplus are more sensitive than larvae to solvents such as acetone, ethanol, ethyl acetate, methanol, triton and xylol (xylene) and attributed this to differences in the composition of the larval and adult cuticle. In the present study, however, adult engorged female R. microplus were more resistant to the hexane extract than the larvae. The waxy layer only occurs after nymph ecdysis and is most pronounced in adult ticks, and therefore, toxic chemicals can be sequestered within the wax, hampering their toxic action (Odhiambo, 1982). A toxic physical effect through cuticle solubilization by the essential oil cannot be excluded, although terpenes and phenylpropanoids are known to act on the octopaminergic receptor which acts as

271

a neurotransmitter, neurohormone and neuromodulator in invertebrates (Price and Berry, 2006). The data presented here showed that some extracts of P. aduncum leaves are efficient for the control of R. microplus, yielding similar results to those reported in the literature for a variety of other synthetic and natural agents (Table 4). Particularly, the control of reproduction observed for the P. aduncum ethyl acetate and hexane extracts compare favorably with the data listed form bibliography. However, the results gathered in the table were obtained using different concentrations of compounds, and the bioassay used to study the bioactivity of the compounds is not the same in each case. Nevertheless, Table 4 allows a first comparison between literature reports. It is noticeable that there is no isolated chemical reported as 100% effective for the engorged female control. Essential oils from Eucalyptus staigeriana and Carapa guianensis were effective at relatively high concentrations. Synthetic chemicals were ineffective, showing the importance of new studies facing the discovery of potential control methods exploring biodiversity. Nevertheless some extracts, essential oils or isolated natural chemicals listed in Table 4 were highly toxic for larvae. Particularly, the activity data range of P. aduncum extracts described in the present work is similar to the activity of synthetic acaricides against larvae. Therefore, either the P. aduncum hexane extract or its essential oil could be potential bioinsecticides for the control of R. microplus. The plant is widely distributed in Brazil, it occurs naturally and in great abundance in the Amazon region (Maia et al., 2001) and has potential for its economic exploitation (Gaia et al., 2004). Piper species are widely used in Brazilian folk medicine due to the anti-microbial properties of their constituents (Bastos and Albuquerque, 2004). Compared with synthetic chemicals, one of the main advantages of using P. aduncum extracts for acarid control is that the different constituents of plant extracts can act synergistically, potentiating the biological activity of the plant, and also decreasing the capacity of the ticks to become resistant to the extract (Mgbojikwe and Okoye, 2001). Furthermore, compared to fungal methods of tick control, P. aduncum extracts act more rapidly because there is no incubation period and the fungus needs a favorable host microenvironment in order to complete its life-cycle (Alves, 1998). The three extracts tested in this study showed no statistically differences on the biological properties evaluated on engorged female ticks, but the extracts achieved a 40–62% CR. However, the hexane extract was very toxic for larvae. Further, it was established that the volatile oil in the extract was highly toxic for larvae at all the concentrations tested. The volatile oil is almost pure dillapiole. This shiquimate is an interesting chemical for further studies on developing new strategies for tick control as 100% of mortality was achieved at the micromolar (mM) range. The differences in toxicity between engorged females and larvae could be due to the differences in the composition of the larval and engorged female cuticle, making the larvae more sensitive than the engorged female. More research on the fate, persistence and general toxicity of P. aduncum extracts

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Table 4 Percentage mortality (%M) for Rhipicephalus microplus engorged females (%MF), larvae (%ML) and percentage control of reproduction (%CR) exposed to different materials in studies reported in scientific publications. Material Essential oils Eucalyptus staigeriana Cymbopongo winterianus Carapa guianensis Copaifera reticulate Drimys brasiliensis Piper aduncum Plant simple extracts Azadirachta indica Artocarpus altilis Adenium obesum Tecoma stans Melia azedarach Dahlstedtia pentaphylla Methyl salicylate Calea serrata Curcuma longa Piper aduncum Microorganisms Metarhizium flavoviride Metarhizium anisopliae Metarhizium anisopliae Metarhizium anisopliae Beauveria amorpha Beauveria bassiana Synthetic chemicals d(+)-Limonene Cymiazol Coumaphos Amitraz Deltametrina Piretro Cipermetrina + clorpirifo´s Thymol Amitraz Organic solvents Acetone 100% Acetone 100% Methanol 100% Methanol 100% Ethanol 100% Ethanol 100% Surfactant agents 1% DMSO 1% Tween-80 5% Triton X-100 a

%MF

%ML

100 50 100

100 50

%CR

Chagas et al. (2002a) Martins (2006) Farias et al. (2007) Fernandes and Freitas (2007) Ribeiro et al. (2008a) Data from the study described in the present paper.

1.57a 100 100

10 89.50 73.82a 50 34

10 22

93.92 9.76 100 70.42

50 75.10 46 79.73

61.81

60.02 90.50 96.90 91.20 86.54 83.42

32.40 29.20

17 73.50

98

58.69 89.70 30.95

36.50 28.24 98.60 100 34.60

100 100 45.3 15 14.2 56

0 4.9 2.3

Reference

10 8.9 0 15.4 0 3.9

Chungsamarnyart et al. (1991) Williams (1993) Mgbojikwe and Okoye (2001) Mendes et al. (2002) Borges et al. (2003) Pereira and Famadas (2004) Silveira Novelino et al. (2007) Ribeiro et al. (2008b) Srivastava et al. (in press) Data from the study described in the present paper.

Onofre et al. (2001) Basso et al. (2005) Bahiense et al. (2006) Alonso-Dı´az et al. (2007) Campos et al. (2005) Campos et al. (2005)

Chungsamarnyart and Jansawan, 1996 Vargas et al. (2003) Davey et al. (2003) Campos Ju´nior and Oliveira (2005) Bahiense et al. (2006) Pereira (2006) Furlong et al. (2007) Silveira Novelino et al. (2007) Davey et al. (2008)

Gonc¸alves et Chagas et al. Gonc¸alves et Chagas et al. Gonc¸alves et Chagas et al.

5 0 3

al. (2007) (2003) al. (2007) (2003) al. (2007) (2003)

Gonc¸alves et al. (2007) Gonc¸alves et al. (2007) Gonc¸alves et al. (2007)

Lethality given in the original paper as the lethal concentration 50% (LC50) in mg ml1.

should be performed, but they appear as promising bioinsecticides for tick control. 5. Conclusions We have shown that the hexane extract and the essential oil from P. aduncum were toxic for both larval and adult R. microplus. They could be an alternative to synthetic acaricides for tick control. Particularly, the volatile oil is a promising bioinsecticide, due to its toxicity for larvae at a micro molar range. On the other hand, dillapiole is an interesting lead for the developing of new compounds for tick control.

Acknowledgments The authors thank the Centre of Biotechnology for the Amazon (CBA) and the National Institute for Research in the Amazon (INPA), the Deside´rio Finamor Veterinary Research Institute, the Biotechnology Institute of University of Caxias do Sul, the University of the Republic of the Uruguay and Coordination of Improvement of Higher Education Staff (CAPES) for financial support and collaboration in developing this work; and Dr. Valerie Dee for help with English revision.

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