Parasitol Res DOI 10.1007/s00436-013-3643-5
ORIGINAL PAPER
Entomopathogenic nematodes associated with essential oil of Lippia sidoides for control of Rhipicephalus microplus (Acari: Ixodidae) Caio Márcio Oliveira Monteiro & Laryssa Xavier Araújo & Geovany Amorim Gomes & Tatiane Oliveira Souza Senra & Fernanda Calmon & Erik Daemon & Mario Geraldo de Carvalho & Vânia Rita Elias Pinheiro Bittencourt & John Furlong & Márcia Cristina de Azevedo Prata
Received: 28 September 2013 / Accepted: 9 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract In vitro effect of the association of the entomopathogenic nematodes Heterorhabditis bacteriophora HP88 and Heterorhabditis indica LPP1 with the essential oil of Lippia sidoides on engorged females of Rhipicephalus microplus was assessed. Engorged females with homogeneous weights (p >0.05) were divided into six groups of ten ticks each (each female = an experimental unit). In the treated groups, the ticks were exposed to the oil (40 μl/ml=4 %) and nematodes (300 entomopathogenic nematodes (EPNs/tick) separately and also to the nematodes together with the oil, while in the control group, the females were immersed in Tween 80 (3 %). All the C. M. O. Monteiro (*) : V. R. E. P. Bittencourt Departamento de Parasitologia, Programa de Pós-Graduação em Ciências Veterinárias, Universidade Federal Rural do Rio de Janeiro, 23890-000 Seropedica, Rio de Janeiro, Brazil e-mail:
[email protected] G. A. Gomes : M. G. de Carvalho Departamento de Química, Instituto de Ciências Exatas, Universidade Federal Rural do Rio de Janeiro, 23890-000 Seropedica, Rio de Janeiro, Brazil L. X. Araújo Universidade Federal de Juiz de Fora, Juiz de Fora 36036-900, Minas Gerais, Brazil T. O. S. Senra : F. Calmon : E. Daemon Departamento de Zoologia, Programa de Pós-Graduação em Comportamento e Biologia Animal, Universidade Federal de Juiz de Fora, 36036-900 Juiz de Fora, Minas Gerais, Brazil J. Furlong Embrapa Gado de Leite, 610 Bairro Dom Bosco, 36038-330 Juiz de Fora, Minas Gerais, Brazil M. C. d. A. Prata Embrapa Gado de Leite, Rua Eugênio do Nascimento, 610 Bairro Dom Bosco, 36038-330 Juiz de Fora, Minas Gerais, Brazil
treatments caused a significant reduction in the egg mass weight (p 0.05) were divided into six groups, each containing ten ticks
L. sidoides essential oil The essential oil from L. sidoides leaves was purchased from PRONAT (Produtos Naturais Ltda., Horizonte, Ceará, Brazil), which was extracted from the cultivated plant by the steam distillation method. To prepare the solution containing the essential oil, 200 μl of oil was pipetted into a test tube containing 5 ml of Tween 80 at 3 %. Then, the content was vortexed to homogenize the solution. Analysis of the constituents of the essential oil of L. sidoides This oil sample was previously characterized the study by Gomes et al. (unpublished data) through analysis in a gas chromatograph coupled with mass spectrometer (GC/MS; Shimadzu QP-2010 Plus) (qualitative) and gas chromatography coupled with flame ionization detector (GC/FID; HP 5890 Series II) (quantitative). The identification of the major constituent was done based on the information obtained from the mentioned analytic methods, together with the data generated by comparison of the nuclear magnetic resonance spectra of hydrogen (NMR 1H, in the Bruker 500MHz spectrometer) of the oil, standard pure thymol (Vetec Química Fina Ltda., Rio de Janeiro, Brazil) and a mixture of oil and thymol. Preparation of the suspensions with nematodes Ten aliquots of 10 μl each were obtained from 20 ml of an aqueous suspension of nematodes stored in a 40-ml cell culture bottle. Then, the mean number of IJs per sample was calculated, and from this mean, the suspensions were adjusted to a concentration of 300 IJs/ml.
Parasitol Res
(each female = experimental unit). In the group treated only with nematodes, each female was placed in a Petri dish (6× 6 cm) previously lined with two filter paper sheets, and then, 1.0 ml of solution was pipetted on the sheets from the suspension at a concentration of 300 EPNs per female. In the group treated with the oil, the females were immersed for 5 min in an oil solution and then were placed onto two filter paper sheets to absorb the excess oil solution, and after then, each female was placed in a Petri dish (6×6 cm) lined with two filter paper sheets moistened with 1.0 ml of distilled water. In the groups treated with the association of EPNs + L. sidoides, the ticks were immersed in the oil solution for 5 min and then were placed onto two filter paper sheets to absorb the excess oil solution, and after which, they were transferred to Petri dishes. Then, 1.0 ml of aqueous suspension containing 300 EPNs was pipetted in each dish. In the control group, the females were immersed for 5 min in Tween 80 (3 %) and then placed individually in Petri dishes lined with two filter paper sheets moistened with distilled water. Therefore, six groups were formed: treated with H. bacteriophora HP88, H. indica LPP1, L. sidoides oil, oil + H. bacteriophora HP88 association, oil + H. indica LPP1 association, and control group. The Petri dishes of all groups were sealed with parafilm for 3 days to keep the paper wet in this period. The experimental groups were kept in a climate-controlled chamber (27±1 °C and relative humidity 80±10 %), and the eggs laid were collected daily until the last tick died. The egg masses from each female were placed in marked 10-ml test tubes, which were sealed with hydrophilic cotton and kept in the chamber under the same temperature and humidity conditions to measure the hatching percentage of larvae. The following biological parameters were evaluated: female weight before oviposition, egg mass weight, and hatching percentage of larvae. These values were then used to calculate the percentage of control (Drummond et al. 1973). Statistical analysis The Biostat version 5.0 program was used for all the statistical analyses. The data were analyzed by the nonparametric Kruskal–Wallis and Student–Newman–Keuls tests (p