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Effect of aqueous extracts of Baccharis trimera on development and hatching of Rhipicephalus microplus (Acaridae) eggs
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ARTICLE IN PRESS Veterinary Parasitology xxx (2013) xxx–xxx
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Short communication
Effect of aqueous extracts of Baccharis trimera on development and hatching of Rhipicephalus microplus (Acaridae) eggs Sirlene Fernandes Lázaro, Leydiana Duarte Fonseca, Ernane Ronie Martins, Neide Judith Faria de Oliveira, Eduardo Robson Duarte ∗ Institute of Agricultural Sciences, Universidade Federal de Minas Gerais, Av Universitária 1000, Universitário, Montes Claros MG 39400-006, Brazil
a r t i c l e
i n f o
Article history: Received 23 May 2012 Received in revised form 5 December 2012 Accepted 13 December 2012 Keywords: Baccharis spp. Cattle tick Natural acaricide Immersion test
a b s t r a c t This study evaluated the effects of aqueous extracts of Baccharis trimera (Less.) DC (Asteraceae), colloquially known as carqueja, on egg production, and hatching rate of larvae of Rhipicephalus microplus. Plant samples were collected in Montes Claros, north of Minas Gerais, Brazil. Adult female ticks were distributed into 24 homogeneous groups of 10. The in vitro test was performed by immersing each group in 10 ml solutions of aqueous extracts at 50, 100, 150, or 200 mg of fresh leaves ml−1 . These concentrations were compared with distilled water as negative control and a commercial product as positive control and the tests were repeated four times. The carqueja extract at concentrations of 150 and 200 mg of fresh leaves ml−1 showed 100% efficacy in inhibiting egg hatching and therefore could have potential as an acaricide. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Infestation by Rhipicephalus (Boophilus) microplus can reduce bovine productivity through anemia associated with blood loss, the release of toxins at the bite location, and transmission of possibly lethal parasitic diseases (Grisi et al., 2002; Campos Júnior and Oliveira, 2005; Mans et al., 2004). The climate in Brazil is suitable for completion of the life cycle and dispersion of the tick in most regions throughout the major part of the year (Magalhães and Lima, 1992; Grisi et al., 2002; Campos Júnior and Oliveira, 2005). Rhipicephalus microplus is the cause of almost 75% of losses associated with bovine ectoparasitism, which totals two billion dollars annually in Brazil (Grisi et al., 2002). The losses can be still higher for Bos taurus and its crossbreeds, which are notoriously susceptible to ticks (Campos Júnior and Oliveira, 2005).
∗ Corresponding author. Tel.: +55 38 2101 7707; fax: +55 38 2101 7703. E-mail address: [email protected] (E.R. Duarte).
Alternatives to conventional acaricides should be evaluated, as the resistance of ticks to synthetic compounds is widespread. This is related to indiscriminate use, which contributes to the selection of resistant populations and reduces the useful life of the available compounds (Campos Júnior and Oliveira, 2005; Clemente et al., 2008; FernandezSala et al., 2012). Developing new drugs is expensive, and it is not always possible to keep pace with the evolution of resistance. Conventional products can also promote contamination of the environment and of food for human consumption (Clemente et al., 2008; Santos et al., 2009). Therefore, research on alternative methods of control with plant substances as potential acaricides is of scientific and practical relevance (Prates et al., 1993; Chagas, 2004; Fernandes et al., 2005). Effective plant extracts could reduce drug resistant tick populations, preserving commercial acaricides (Chagas, 2004). The genus Baccharis comprises 150 species, colloquially known as carqueja, bacárida, and bacórida cacália (Lorenzi and Abreu, 2008). Allelopathic, analgesic, anti-fungal, antiinflammatory, antimicrobial, anti-mutagenic, anti-oxidant, anti-viral, and insecticidal actions have been described in these species (Nakasugi and Komai, 1998; Abad et al.,
0304-4017/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.12.025
Please cite this article in press as: Lázaro, S.F., et al., Effect of aqueous extracts of Baccharis trimera on development and hatching of Rhipicephalus microplus (Acaridae) eggs. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2012.12.025
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Table 1 Mean egg mass (g), number of hatched larvae (n), and the reproductive efficacy of cattle tick treated with varying concentrations of Baccharis trimera aqueous extract. These are compared with efficacy using distilled water or commercial acaricide (12.5 mg l−1 cypermethrin combined with 150 mg l−1 dichlorvos). Treatments
Eggs mass (g)
Baccharis trimera 50 mg ml−1 100 mg ml−1 150 mg ml−1 200 mg ml−1 Positive controlb Negative control Variation coefficient (%)
0.55 ab 0.53 ab 0.47 ab 0.42 bb 0.24 cc 0.61 aa 20.51
Hatched larvae, n 815.00 ab 749.00 ab 0c 0c 20.00 c 2,246.63 a 37.32
Reproductive efficacy
Effectiveness (%)a
38,389.96 35,580.08 0.00 0.00 941.16 109,355.79
64.89 67.47 100.00 100.00 99.14 0.00
Means followed by same letter in columns are similar statistically by Duncan’s test at 5% probability (P ≤ 0.05). a Means obtained by the equation from Drummond et al. (1973). b Commercial acaricide, containing 12.5 mg ml−1 cypermethrin and 150 mg ml−1 dichlorvos-phosphate.
1999; Feresin et al., 2003; Paul et al., 2009). The objective of this study was to investigate the effects of carqueja aqueous extract (CAE) to egg production and hatching of R. microplus. 2. Materials and methods The plant was grown in the medicinal garden of the Institute of Agricultural Sciences, Federal University of Minas Gerais (ICA/UFMG), Montes Claros, Brazil. The plants were collected in July and August 2010, and cataloged in the herbarium of this institution. Fresh leaves were chopped into approximately 2 cm2 pieces, and the aqueous extract was prepared by adding 20 g of fresh leaves to 100 ml boiling distilled water, according to the method by Siqueira (1956). This mixture was placed in a beaker and maintained at boiling point for 15 min. The initial volume was maintained by adding sterile distilled water, followed by hot filtration through a cotton and gauze funnel. The CAE at a concentration equivalent to 200 mg of fresh leaves ml−1 was diluted with sterile distilled water, to make 150, 100, 50 mg ml−1 solutions. Engorged adult females of R. microplus were collected from naturally infested Holstein cows in Bocaiúva city of Minas Gerais State, Brazil. Ticks were packed in aerated plastic containers and sent to the ICA/UFMG. Females larger than 4 mm were selected, washed with distilled water, placed on paper towels, and divided into 24 homogeneous groups of 10 females, based on the degree of engorgement and weight. Sterile distilled water was used as negative control, and a commercial product containing the mixture of 12.5 mg ml−1 of cypermethrin and 150 mg ml−1 of dichlorvos-phosphate (Flytick Plus, Valleé S.A., São Paulo, SP, Brazil), diluted as recommended by the manufacturer, was the positive control. This commercial product was selected because it was successfully used for tick control in the farm. The collection of engorged female ticks was conducted at the beginning of the rainy season between December and February after at least 60 days of last acaricide application. The acaricidal effectiveness was evaluated by an immersion test according to Drummond et al. (1973). The 24 groups were immersed in 10 ml of test solution for 5 min. Excess solution was removed with a paper towel, and ticks were placed in a Petri dish and maintained at 28 ◦ C and
70% relative humidity in an environmental chamber BOD incubator. Fifteen days after the initiation of tick egg laying, the mass of eggs for each group was determined on an analytical scale and transferred to 3 ml plastic syringes. Thirty days after the start of hatching, the syringe contents were transferred to Petri dishes, and the larvae and eggs were counted under a stereoscopic microscope to determine the hatching rate in each group. The efficacy of treatment (product efficacy) was estimated using the equation of Drummond et al. (1973): ER (reproductive efficacy) =
Egg weight × % hatching × 20, 000∗ Tick weight
* Constant
of 20,000 eggs g−1 of mass.
EP (efficacy of product) =
(ER control group − ER treated group) × 100 ER control group
A randomized design was used to compare the four carqueja concentrations with distilled water control and a commercial acaricide control and the tests were repeated four times. The data were log transformed (x + 10) and subjected to analysis of variance. The means were compared by Duncan’s test (P ≤ 0.05). The concentration of the extract sufficient to inhibit 90% of the hatching (LC90 ) was calculated by probit analysis using the statistical package Saeg 9.1 (Saeg, 2007). 3. Results and discussion The average egg mass produced by ticks treated with 200 mg ml−1 of CAE was significantly lower than that of the negative control with distilled water (P ≤ 0.05). There was no hatching at concentrations of 150 and 200 mg ml−1 (Table 1). Thus, carqueja aqueous extract at these concentrations produced 100% inhibition of egg hatching, higher than the 95% minimum efficacy required for conventional acaricides (Brasil, 1990). The LC90 of CAE on hatching inhibition of R. microplus, estimated by probit analysis was 121.84 mg of fresh leaves ml−1 (Fig. 1).
Please cite this article in press as: Lázaro, S.F., et al., Effect of aqueous extracts of Baccharis trimera on development and hatching of Rhipicephalus microplus (Acaridae) eggs. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2012.12.025
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Fig. 1. Percent egg hatch from Rhipicephalus microplus exposed to varying concentrations of aqueous extract of Baccharis trimera (carqueja).
Other studies with aqueous plant extracts have also evidenced acaricide effectiveness. A preview research evaluated the effect of aqueous extract of Calotropis procera Ait. (R. Br.) on the reproductive performance of R. microplus. The acaricide efficacy at 5 and 25% concentrations of this plant extract was higher than obtained for a commercial product, which corresponded to 93.98% (Lázaro et al., 2012). In other study, aqueous extract of Simarouba versicolor (St. Hill) was evaluated about the oviposition of the cattle tick. The LC50 was 4.14 mg ml−1 and the 17.2 mg ml−1 concentration was capable of inhibiting 100% of the oviposition (Pires et al., 2007). Acaricidal activity of aqueous leaf extract of Manilkara zapota was carried out against R. microplus and the results showed the LC50 values of 16.72 mg ml−1 (Rajakumar and Abdul Rahuman, 2012), lower than LC50 observed to CAE (45.31 mg ml−1 ) in this present study that was represented by mg of fresh leaves ml−1 . The research of effectiveness of aqueous extract of Azadiracta indica on the control of bovine ticks during a month showed no significant difference between bovine groups topically treated with neem extract or abamectin. The average of engorged female ticks per animal at 30 day was significantly lower than at zero and 15 days (Valente et al., 2007). Phitochemical analysis showed that alkaloids from A. indica can reduce oviposition and fecundity of R. microplus (Silva et al., 2007). Alkaloid metabolites have also been identified in hydroalcohol or aqueous extracts from Baccharis trimera (Mendes et al., 2007) and may be active components producing the results obtained in this present study, a topic for further investigation. The results of this study are promising for the use of CAE as an acaricide in cattle, especially in Brazil where the climate allows the reproduction of R. microplus most of the year and where the this plant is easily found (Magalhães and Lima, 1992; Campos Júnior and Oliveira, 2005). As the damage related to R. microplus in cattle represents billions of dollars in economic losses annually (Grisi et al., 2002; Campos Júnior and Oliveira, 2005), the evident acaricidal
effect of CAE should be considered in its control. The results support the importance and diversity of biological actions described for this plant (Nakasugi and Komai, 1998; Abad et al., 1999; Feresin et al., 2003; Paul et al., 2009) and provide a potential alternative to commercial acaricides (Prates et al., 1993; Campos Júnior and Oliveira, 2005; Fernandes et al., 2005). The CAE is easily prepared. Following determination of its toxicity, safety and residue depletion profile, it could reduce contamination of the environment and foods of animal origin and avoid the disposal of milk from treated cattle required with synthetic products (Clemente et al., 2008; Santos et al., 2009). The CAE at 150 mg of fresh leaves ml−1 showed high acaricidal efficacy, through inhibiting egg hatching. Toxicological tests and analyses in vivo are essential to determine appropriate dosages and frequency of application necessary to promote carqueja extract as an alternative acaricide. Conflict of interest statement The authors of this manuscript have no financial or personal relationship with people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgments The authors thank the Coordination of Improvement of Higher Education Personnel (CAPES), the National Council for Scientific and Technological Development (CNPq), Foundation for Research Support of Minas Gerais (FAPEMIG), and the Tutorial Education ProgramDepartment of Higher Education (PET-SESU). References Abad, M.J., Bermejo, P., Gonzáles, E., Iglesias, I., Irurzum, A., Carrasco, L., 1999. Antiviral activity of Bolivian plant extracts. Gen. Pharmacol. 32, 499–503.
Please cite this article in press as: Lázaro, S.F., et al., Effect of aqueous extracts of Baccharis trimera on development and hatching of Rhipicephalus microplus (Acaridae) eggs. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2012.12.025
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Brasil, 1990. Ministério da Agricultura. Portaria no. 90, December 4, 1990. Normas para produc¸ão, controle e utilizac¸ão de produtos antiparasitários. Diário Oficial, sec 1, col. 2. Campos Júnior, D.A., Oliveira, P.R., 2005. Avaliac¸ão in vitro da eficácia de acaricidas sobre Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae) de bovinos no município de Ilhéus, Bahia, Brasil. Cienc. Rural 35, 1386–1392. Chagas, A.C.S., 2004. Controle de parasitos utilizando extratos de plantas. Rev. Bras. Parasitol. Vet. 13, 156–160. Clemente, M.A., Gomes, F.G., Scotton, A.C.B.S., Goldner, M.S., Reis, E.S., Almeida, M.N., 2008. Avaliac¸ão do potencial de plantas medicinais no controle de Boophilus microplus (Acari: Ixodidae). Rev. Bras. Biociênc. 5, 516–518. 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. J. Econ. Entomol. 66, 130–133. Feresin, G.E., Tapia, A., Gimenez, A., Ravelo, A.G., Zacchino, S., Sortino, M., Schmeda-Hirschmann, G., 2003. Constituents of the Argentinian medicinal plant Baccharis grisebachii and their antimicrobial activity. J. Ethnopharmacol. 89, 73–80. Fernandes, F.F., Freitas, E.P.S., Costa, A.C., Silva, I.G., 2005. Potencial larvicida de Sapindus saponaria para o controle do carrapato bovino Boophilus microplus. Pesq. Agropec. Bras. 40, 1243–1245. Fernandez-Sala, A., Rodriguez-Vivas, R.I., Alonso-Dia, M.A., 2012. First report of a Rhipicephalus microplus tick population multi-resistant to acaricides and ivermectin in the Mexican tropics. Vet. Parasitol. 183, 338–342. Grisi, L., Massard, C., Moya-Borja, G.E., Pereira, J.B., 2002. Impáctos econômicos das principais infestac¸ões em bovines no Brasil. Hora Veter. 21, 8–10. Lázaro, S.F., Fonseca, L.D., Fernandes, R.C., Tolentino, J.S., Martins, E.R., Duarte, E.R., 2012. Efeitos do extrato aquoso do algodão de seda (Calotropis procera Ait, R. Br.) na performance reprodutiva de Rhipicephalus microplus. Rev. Bras. Plantas Med. 14, 302–305. Lorenzi, H.M., Abreu, F.J. (Eds.), 2008. Plantas medicinais no Brasil: nativas e exóticas. , 2nd ed. Instituto Plantarum, Nova Odessa, 576 pp. Magalhães, F.E.P., Lima, J.D., 1992. Desenvolvimento e sobrevivência do carrapato bovino na pastagem Brachiaria decumbens em Pedro Leopoldo, MG, Brasil. Pesq. Agropec. Bras. 27, 15–25. Mans, R., Gothe, A., Neitz, W.H., 2004. Biochemical perspectives on paralysis and other forms of toxicoses caused by ticks. Parasitology 129, 95–111.
Mendes, F.R., Tabach, R., Carlini, E.A., 2007. Evaluation of Baccharis trimera and Davilla rugosa in tests for adaptogen activity. Phytother. Res. 6, 517–522. Nakasugi, T., Komai, K., 1998. Antimutagens in the Brazilian folk medicinal plant carqueja (Baccharis trimera Less.). J. Agric. Food Chem. 46, 2560–2564. Paul, E.L., Lunardelli, A., Caberlon, E., Oliveira, C.B., Santos, R.C.V., Biolchi, V., Bastos, C.M.A., Moreira, K.B., Nunes, F.B., Gosmann, G., Oliveira, J.R., 2009. Anti-inflammatory and immunomodulatory effects of Baccharis trimera aqueous extract on induced pleurisy in rats and lymphoproliferation in vitro. Inflammation 32, 419–425. Pires, J.E.P., Fernandes, R.M., Fernandes, M.Z.L., Viana, G.E.N., Dourado, J.C.L., Sousa, S.A.A., 2007. Determinac¸ão da concentrac¸ão inibitória média (CI50) do extrato aquoso de Simarouba versicolor, St. Hill sobre a ovipostura do carrapato bovino (Boophilus microplus, Canestrine, 1887). Rev. Bras. Plantas Med. 9, 23–26. Prates, H.T., Oliveira, A.B., Leite, R.C., Craveiro, A.A., 1993. Anti-tick effect and chemical composition of the molasses Grass essential oil. Pesq. Agropec. Bras. 28, 621–625. Rajakumar, G., Abdul Rahuman, A., 2012. Acaricidal activity of aqueous extract and synthesized silver nanoparticles from Manilkara zapota against Rhipicephalus (Boophilus) microplus. Res. Vet. Sci. 93, 303–309. Saeg, 2007. Sistema para Análises Estatísticas, Versão 9.1. Fundac¸ão Arthur Bernardes – UFV, Vic¸osa. Santos, T.R.B., Farias, N.A.R., Cunha Filho, N.A., Pappen, F.G., Vaz Junior, I.S., 2009. Abordagem sobre o controle do carrapato Rhipicephalus (Boophilus) microplus no sul do Rio Grande do Sul. Pesq. Vet. Bras. 29, 65–70. Silva, W.W., Athayde, A.C.R., Rodrigues, O.G., Araújo, G.M.B., Santos, V.D., Neto, A.B.S., Coelho, M.C.O.C., Marinho, M.L., 2007. Efeitos do neem (Azadirachta indica A, Juss) e do capim santo [Cymbopogon citratus (DC) Stapf] sobre os parâmetros reprodutivos de fêmeas ingurgitadas de Boophilus microplus e Rhipicephalus sanguineus (Acari: Ixodidae) no semiárido paraibano. Rev. Bras. Plantas Med. 9, 1–5. Siqueira, S.A. (Ed.), 1956. Farmacopéia dos Estados Unidos do Brasil. , 2nd ed. Indústria Gráfica Siqueira, São Paulo, 1265 pp. Valente, M., Barranco, A., Sellaive-Villaroel, A.B., 2007. Effectiveness of Azadiracta indica watery extract on the control of bovine ticks. Arq. Bras. Med. Vet. Zootec. 59, 1341–1343.
Please cite this article in press as: Lázaro, S.F., et al., Effect of aqueous extracts of Baccharis trimera on development and hatching of Rhipicephalus microplus (Acaridae) eggs. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2012.12.025
ARTICLE IN PRESS Veterinary Parasitology xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Effect of aqueous extracts of Baccharis trimera on development and hatching of Rhipicephalus microplus (Acaridae) eggs Sirlene Fernandes Lázaro, Leydiana Duarte Fonseca, Ernane Ronie Martins, Neide Judith Faria de Oliveira, Eduardo Robson Duarte ∗ Institute of Agricultural Sciences, Universidade Federal de Minas Gerais, Av Universitária 1000, Universitário, Montes Claros MG 39400-006, Brazil
a r t i c l e
i n f o
Article history: Received 23 May 2012 Received in revised form 5 December 2012 Accepted 13 December 2012 Keywords: Baccharis spp. Cattle tick Natural acaricide Immersion test
a b s t r a c t This study evaluated the effects of aqueous extracts of Baccharis trimera (Less.) DC (Asteraceae), colloquially known as carqueja, on egg production, and hatching rate of larvae of Rhipicephalus microplus. Plant samples were collected in Montes Claros, north of Minas Gerais, Brazil. Adult female ticks were distributed into 24 homogeneous groups of 10. The in vitro test was performed by immersing each group in 10 ml solutions of aqueous extracts at 50, 100, 150, or 200 mg of fresh leaves ml−1 . These concentrations were compared with distilled water as negative control and a commercial product as positive control and the tests were repeated four times. The carqueja extract at concentrations of 150 and 200 mg of fresh leaves ml−1 showed 100% efficacy in inhibiting egg hatching and therefore could have potential as an acaricide. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Infestation by Rhipicephalus (Boophilus) microplus can reduce bovine productivity through anemia associated with blood loss, the release of toxins at the bite location, and transmission of possibly lethal parasitic diseases (Grisi et al., 2002; Campos Júnior and Oliveira, 2005; Mans et al., 2004). The climate in Brazil is suitable for completion of the life cycle and dispersion of the tick in most regions throughout the major part of the year (Magalhães and Lima, 1992; Grisi et al., 2002; Campos Júnior and Oliveira, 2005). Rhipicephalus microplus is the cause of almost 75% of losses associated with bovine ectoparasitism, which totals two billion dollars annually in Brazil (Grisi et al., 2002). The losses can be still higher for Bos taurus and its crossbreeds, which are notoriously susceptible to ticks (Campos Júnior and Oliveira, 2005).
∗ Corresponding author. Tel.: +55 38 2101 7707; fax: +55 38 2101 7703. E-mail address: [email protected] (E.R. Duarte).
Alternatives to conventional acaricides should be evaluated, as the resistance of ticks to synthetic compounds is widespread. This is related to indiscriminate use, which contributes to the selection of resistant populations and reduces the useful life of the available compounds (Campos Júnior and Oliveira, 2005; Clemente et al., 2008; FernandezSala et al., 2012). Developing new drugs is expensive, and it is not always possible to keep pace with the evolution of resistance. Conventional products can also promote contamination of the environment and of food for human consumption (Clemente et al., 2008; Santos et al., 2009). Therefore, research on alternative methods of control with plant substances as potential acaricides is of scientific and practical relevance (Prates et al., 1993; Chagas, 2004; Fernandes et al., 2005). Effective plant extracts could reduce drug resistant tick populations, preserving commercial acaricides (Chagas, 2004). The genus Baccharis comprises 150 species, colloquially known as carqueja, bacárida, and bacórida cacália (Lorenzi and Abreu, 2008). Allelopathic, analgesic, anti-fungal, antiinflammatory, antimicrobial, anti-mutagenic, anti-oxidant, anti-viral, and insecticidal actions have been described in these species (Nakasugi and Komai, 1998; Abad et al.,
0304-4017/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.12.025
Please cite this article in press as: Lázaro, S.F., et al., Effect of aqueous extracts of Baccharis trimera on development and hatching of Rhipicephalus microplus (Acaridae) eggs. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2012.12.025
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Table 1 Mean egg mass (g), number of hatched larvae (n), and the reproductive efficacy of cattle tick treated with varying concentrations of Baccharis trimera aqueous extract. These are compared with efficacy using distilled water or commercial acaricide (12.5 mg l−1 cypermethrin combined with 150 mg l−1 dichlorvos). Treatments
Eggs mass (g)
Baccharis trimera 50 mg ml−1 100 mg ml−1 150 mg ml−1 200 mg ml−1 Positive controlb Negative control Variation coefficient (%)
0.55 ab 0.53 ab 0.47 ab 0.42 bb 0.24 cc 0.61 aa 20.51
Hatched larvae, n 815.00 ab 749.00 ab 0c 0c 20.00 c 2,246.63 a 37.32
Reproductive efficacy
Effectiveness (%)a
38,389.96 35,580.08 0.00 0.00 941.16 109,355.79
64.89 67.47 100.00 100.00 99.14 0.00
Means followed by same letter in columns are similar statistically by Duncan’s test at 5% probability (P ≤ 0.05). a Means obtained by the equation from Drummond et al. (1973). b Commercial acaricide, containing 12.5 mg ml−1 cypermethrin and 150 mg ml−1 dichlorvos-phosphate.
1999; Feresin et al., 2003; Paul et al., 2009). The objective of this study was to investigate the effects of carqueja aqueous extract (CAE) to egg production and hatching of R. microplus. 2. Materials and methods The plant was grown in the medicinal garden of the Institute of Agricultural Sciences, Federal University of Minas Gerais (ICA/UFMG), Montes Claros, Brazil. The plants were collected in July and August 2010, and cataloged in the herbarium of this institution. Fresh leaves were chopped into approximately 2 cm2 pieces, and the aqueous extract was prepared by adding 20 g of fresh leaves to 100 ml boiling distilled water, according to the method by Siqueira (1956). This mixture was placed in a beaker and maintained at boiling point for 15 min. The initial volume was maintained by adding sterile distilled water, followed by hot filtration through a cotton and gauze funnel. The CAE at a concentration equivalent to 200 mg of fresh leaves ml−1 was diluted with sterile distilled water, to make 150, 100, 50 mg ml−1 solutions. Engorged adult females of R. microplus were collected from naturally infested Holstein cows in Bocaiúva city of Minas Gerais State, Brazil. Ticks were packed in aerated plastic containers and sent to the ICA/UFMG. Females larger than 4 mm were selected, washed with distilled water, placed on paper towels, and divided into 24 homogeneous groups of 10 females, based on the degree of engorgement and weight. Sterile distilled water was used as negative control, and a commercial product containing the mixture of 12.5 mg ml−1 of cypermethrin and 150 mg ml−1 of dichlorvos-phosphate (Flytick Plus, Valleé S.A., São Paulo, SP, Brazil), diluted as recommended by the manufacturer, was the positive control. This commercial product was selected because it was successfully used for tick control in the farm. The collection of engorged female ticks was conducted at the beginning of the rainy season between December and February after at least 60 days of last acaricide application. The acaricidal effectiveness was evaluated by an immersion test according to Drummond et al. (1973). The 24 groups were immersed in 10 ml of test solution for 5 min. Excess solution was removed with a paper towel, and ticks were placed in a Petri dish and maintained at 28 ◦ C and
70% relative humidity in an environmental chamber BOD incubator. Fifteen days after the initiation of tick egg laying, the mass of eggs for each group was determined on an analytical scale and transferred to 3 ml plastic syringes. Thirty days after the start of hatching, the syringe contents were transferred to Petri dishes, and the larvae and eggs were counted under a stereoscopic microscope to determine the hatching rate in each group. The efficacy of treatment (product efficacy) was estimated using the equation of Drummond et al. (1973): ER (reproductive efficacy) =
Egg weight × % hatching × 20, 000∗ Tick weight
* Constant
of 20,000 eggs g−1 of mass.
EP (efficacy of product) =
(ER control group − ER treated group) × 100 ER control group
A randomized design was used to compare the four carqueja concentrations with distilled water control and a commercial acaricide control and the tests were repeated four times. The data were log transformed (x + 10) and subjected to analysis of variance. The means were compared by Duncan’s test (P ≤ 0.05). The concentration of the extract sufficient to inhibit 90% of the hatching (LC90 ) was calculated by probit analysis using the statistical package Saeg 9.1 (Saeg, 2007). 3. Results and discussion The average egg mass produced by ticks treated with 200 mg ml−1 of CAE was significantly lower than that of the negative control with distilled water (P ≤ 0.05). There was no hatching at concentrations of 150 and 200 mg ml−1 (Table 1). Thus, carqueja aqueous extract at these concentrations produced 100% inhibition of egg hatching, higher than the 95% minimum efficacy required for conventional acaricides (Brasil, 1990). The LC90 of CAE on hatching inhibition of R. microplus, estimated by probit analysis was 121.84 mg of fresh leaves ml−1 (Fig. 1).
Please cite this article in press as: Lázaro, S.F., et al., Effect of aqueous extracts of Baccharis trimera on development and hatching of Rhipicephalus microplus (Acaridae) eggs. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2012.12.025
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Fig. 1. Percent egg hatch from Rhipicephalus microplus exposed to varying concentrations of aqueous extract of Baccharis trimera (carqueja).
Other studies with aqueous plant extracts have also evidenced acaricide effectiveness. A preview research evaluated the effect of aqueous extract of Calotropis procera Ait. (R. Br.) on the reproductive performance of R. microplus. The acaricide efficacy at 5 and 25% concentrations of this plant extract was higher than obtained for a commercial product, which corresponded to 93.98% (Lázaro et al., 2012). In other study, aqueous extract of Simarouba versicolor (St. Hill) was evaluated about the oviposition of the cattle tick. The LC50 was 4.14 mg ml−1 and the 17.2 mg ml−1 concentration was capable of inhibiting 100% of the oviposition (Pires et al., 2007). Acaricidal activity of aqueous leaf extract of Manilkara zapota was carried out against R. microplus and the results showed the LC50 values of 16.72 mg ml−1 (Rajakumar and Abdul Rahuman, 2012), lower than LC50 observed to CAE (45.31 mg ml−1 ) in this present study that was represented by mg of fresh leaves ml−1 . The research of effectiveness of aqueous extract of Azadiracta indica on the control of bovine ticks during a month showed no significant difference between bovine groups topically treated with neem extract or abamectin. The average of engorged female ticks per animal at 30 day was significantly lower than at zero and 15 days (Valente et al., 2007). Phitochemical analysis showed that alkaloids from A. indica can reduce oviposition and fecundity of R. microplus (Silva et al., 2007). Alkaloid metabolites have also been identified in hydroalcohol or aqueous extracts from Baccharis trimera (Mendes et al., 2007) and may be active components producing the results obtained in this present study, a topic for further investigation. The results of this study are promising for the use of CAE as an acaricide in cattle, especially in Brazil where the climate allows the reproduction of R. microplus most of the year and where the this plant is easily found (Magalhães and Lima, 1992; Campos Júnior and Oliveira, 2005). As the damage related to R. microplus in cattle represents billions of dollars in economic losses annually (Grisi et al., 2002; Campos Júnior and Oliveira, 2005), the evident acaricidal
effect of CAE should be considered in its control. The results support the importance and diversity of biological actions described for this plant (Nakasugi and Komai, 1998; Abad et al., 1999; Feresin et al., 2003; Paul et al., 2009) and provide a potential alternative to commercial acaricides (Prates et al., 1993; Campos Júnior and Oliveira, 2005; Fernandes et al., 2005). The CAE is easily prepared. Following determination of its toxicity, safety and residue depletion profile, it could reduce contamination of the environment and foods of animal origin and avoid the disposal of milk from treated cattle required with synthetic products (Clemente et al., 2008; Santos et al., 2009). The CAE at 150 mg of fresh leaves ml−1 showed high acaricidal efficacy, through inhibiting egg hatching. Toxicological tests and analyses in vivo are essential to determine appropriate dosages and frequency of application necessary to promote carqueja extract as an alternative acaricide. Conflict of interest statement The authors of this manuscript have no financial or personal relationship with people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgments The authors thank the Coordination of Improvement of Higher Education Personnel (CAPES), the National Council for Scientific and Technological Development (CNPq), Foundation for Research Support of Minas Gerais (FAPEMIG), and the Tutorial Education ProgramDepartment of Higher Education (PET-SESU). References Abad, M.J., Bermejo, P., Gonzáles, E., Iglesias, I., Irurzum, A., Carrasco, L., 1999. Antiviral activity of Bolivian plant extracts. Gen. Pharmacol. 32, 499–503.
Please cite this article in press as: Lázaro, S.F., et al., Effect of aqueous extracts of Baccharis trimera on development and hatching of Rhipicephalus microplus (Acaridae) eggs. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2012.12.025
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Brasil, 1990. Ministério da Agricultura. Portaria no. 90, December 4, 1990. Normas para produc¸ão, controle e utilizac¸ão de produtos antiparasitários. Diário Oficial, sec 1, col. 2. Campos Júnior, D.A., Oliveira, P.R., 2005. Avaliac¸ão in vitro da eficácia de acaricidas sobre Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae) de bovinos no município de Ilhéus, Bahia, Brasil. Cienc. Rural 35, 1386–1392. Chagas, A.C.S., 2004. Controle de parasitos utilizando extratos de plantas. Rev. Bras. Parasitol. Vet. 13, 156–160. Clemente, M.A., Gomes, F.G., Scotton, A.C.B.S., Goldner, M.S., Reis, E.S., Almeida, M.N., 2008. Avaliac¸ão do potencial de plantas medicinais no controle de Boophilus microplus (Acari: Ixodidae). Rev. Bras. Biociênc. 5, 516–518. 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. J. Econ. Entomol. 66, 130–133. Feresin, G.E., Tapia, A., Gimenez, A., Ravelo, A.G., Zacchino, S., Sortino, M., Schmeda-Hirschmann, G., 2003. Constituents of the Argentinian medicinal plant Baccharis grisebachii and their antimicrobial activity. J. Ethnopharmacol. 89, 73–80. Fernandes, F.F., Freitas, E.P.S., Costa, A.C., Silva, I.G., 2005. Potencial larvicida de Sapindus saponaria para o controle do carrapato bovino Boophilus microplus. Pesq. Agropec. Bras. 40, 1243–1245. Fernandez-Sala, A., Rodriguez-Vivas, R.I., Alonso-Dia, M.A., 2012. First report of a Rhipicephalus microplus tick population multi-resistant to acaricides and ivermectin in the Mexican tropics. Vet. Parasitol. 183, 338–342. Grisi, L., Massard, C., Moya-Borja, G.E., Pereira, J.B., 2002. Impáctos econômicos das principais infestac¸ões em bovines no Brasil. Hora Veter. 21, 8–10. Lázaro, S.F., Fonseca, L.D., Fernandes, R.C., Tolentino, J.S., Martins, E.R., Duarte, E.R., 2012. Efeitos do extrato aquoso do algodão de seda (Calotropis procera Ait, R. Br.) na performance reprodutiva de Rhipicephalus microplus. Rev. Bras. Plantas Med. 14, 302–305. Lorenzi, H.M., Abreu, F.J. (Eds.), 2008. Plantas medicinais no Brasil: nativas e exóticas. , 2nd ed. Instituto Plantarum, Nova Odessa, 576 pp. Magalhães, F.E.P., Lima, J.D., 1992. Desenvolvimento e sobrevivência do carrapato bovino na pastagem Brachiaria decumbens em Pedro Leopoldo, MG, Brasil. Pesq. Agropec. Bras. 27, 15–25. Mans, R., Gothe, A., Neitz, W.H., 2004. Biochemical perspectives on paralysis and other forms of toxicoses caused by ticks. Parasitology 129, 95–111.
Mendes, F.R., Tabach, R., Carlini, E.A., 2007. Evaluation of Baccharis trimera and Davilla rugosa in tests for adaptogen activity. Phytother. Res. 6, 517–522. Nakasugi, T., Komai, K., 1998. Antimutagens in the Brazilian folk medicinal plant carqueja (Baccharis trimera Less.). J. Agric. Food Chem. 46, 2560–2564. Paul, E.L., Lunardelli, A., Caberlon, E., Oliveira, C.B., Santos, R.C.V., Biolchi, V., Bastos, C.M.A., Moreira, K.B., Nunes, F.B., Gosmann, G., Oliveira, J.R., 2009. Anti-inflammatory and immunomodulatory effects of Baccharis trimera aqueous extract on induced pleurisy in rats and lymphoproliferation in vitro. Inflammation 32, 419–425. Pires, J.E.P., Fernandes, R.M., Fernandes, M.Z.L., Viana, G.E.N., Dourado, J.C.L., Sousa, S.A.A., 2007. Determinac¸ão da concentrac¸ão inibitória média (CI50) do extrato aquoso de Simarouba versicolor, St. Hill sobre a ovipostura do carrapato bovino (Boophilus microplus, Canestrine, 1887). Rev. Bras. Plantas Med. 9, 23–26. Prates, H.T., Oliveira, A.B., Leite, R.C., Craveiro, A.A., 1993. Anti-tick effect and chemical composition of the molasses Grass essential oil. Pesq. Agropec. Bras. 28, 621–625. Rajakumar, G., Abdul Rahuman, A., 2012. Acaricidal activity of aqueous extract and synthesized silver nanoparticles from Manilkara zapota against Rhipicephalus (Boophilus) microplus. Res. Vet. Sci. 93, 303–309. Saeg, 2007. Sistema para Análises Estatísticas, Versão 9.1. Fundac¸ão Arthur Bernardes – UFV, Vic¸osa. Santos, T.R.B., Farias, N.A.R., Cunha Filho, N.A., Pappen, F.G., Vaz Junior, I.S., 2009. Abordagem sobre o controle do carrapato Rhipicephalus (Boophilus) microplus no sul do Rio Grande do Sul. Pesq. Vet. Bras. 29, 65–70. Silva, W.W., Athayde, A.C.R., Rodrigues, O.G., Araújo, G.M.B., Santos, V.D., Neto, A.B.S., Coelho, M.C.O.C., Marinho, M.L., 2007. Efeitos do neem (Azadirachta indica A, Juss) e do capim santo [Cymbopogon citratus (DC) Stapf] sobre os parâmetros reprodutivos de fêmeas ingurgitadas de Boophilus microplus e Rhipicephalus sanguineus (Acari: Ixodidae) no semiárido paraibano. Rev. Bras. Plantas Med. 9, 1–5. Siqueira, S.A. (Ed.), 1956. Farmacopéia dos Estados Unidos do Brasil. , 2nd ed. Indústria Gráfica Siqueira, São Paulo, 1265 pp. Valente, M., Barranco, A., Sellaive-Villaroel, A.B., 2007. Effectiveness of Azadiracta indica watery extract on the control of bovine ticks. Arq. Bras. Med. Vet. Zootec. 59, 1341–1343.
Please cite this article in press as: Lázaro, S.F., et al., Effect of aqueous extracts of Baccharis trimera on development and hatching of Rhipicephalus microplus (Acaridae) eggs. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2012.12.025