In Vitro Evaluation of Essential Oils Derived from Piper nigrum (Piperaceae) and Citrus limonum (Rutaceae) against the Tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).
The tick Rhipicephalus (Boophilus) microplus (Canestrini, 1887) is an ectoparasite of cattle , present in tropical and subtropical areas of America, Africa, Asia, and Australia in latitudes between 32[degrees]N and 32[degrees]S [2, 3]. About the damage caused by this parasite, direct losses in the production of milk and meat, leather damage caused by inflammatory reactions at the sites of tick attachment, appearance of myiasis, and the transmission of diseases such as bovine tick fever (caused by protozoa of the genus Babesia and by bacteria of the genus Anaplasma) could be mentioned . The economic losses of Brazil related to ticks are estimated by 3.24 billion dollars a year, represented by the direct action of the parasite in the animals and by the cost of the control systems .
One of the strategies to minimize the economic losses caused by this pest involves reducing tick populations to economically acceptable levels . But the inherent problems in controlling this parasite are acaricide resistance, and environmental residues resulted in only partial success of tick control . The searches for environmentally safe products have accelerated the research on botanical acaricides [8, 9]. Plants have been used as food, spices, and medicines for thousands of years. These medicines initially took the form of crude drugs such as a variety of plants formulations. Natural products have been the origin of many important molecules in drug discoveries. Different culinary herbs have been screened for their biological activities. The active ingredients from plants are known to possess insecticidal, growth inhibiting, antimoulting, and repellent activities . Thus, there is increasing interest in alternative methods for tick control and growing demand for nonchemical products in livestock that are safer to animals and the environment [10, 11]. Botanical alternatives, such as essential oils, are currently receiving particular attention .
In Brazil, the Rutaceae family comprises about 38 genera and 22 species . Citrus limonum (L.) comprises 3 species and various hybrids. Species of this genus are known to show monoterpenes and sesquiterpenes, such as limonene and apinene [14,15]. Essential oils of this kind have been evaluated for presenting biological activities such as insecticide, cytotoxic, and antibacterial activities .
Piper nigrum (L.), popularly known as black pepper, is a member of family Piperaceae [17, 18]. The genus Piper includes more than 1000 species, but the best known are P nigrum, Piper longum, and Piper betle [19, 20]. The fruits of P nigrum contain piperine, a compound that has already shown leishmanicidal activity . Furthermore, secondary metabolites of this species possess active compounds with insecticidal activity, antibacterial, antifungal, and others [20, 22].
The aim of this work is to develop natural products, like essential oils, for the combat of the cattle tick.
2. Materials and Methods
2.1. Essential Oil. Citrus limonum and Piper nigrum essential oils were obtained commercially in Bellarome, RJ, Brazil commercial Ltda. According to the company, C. limonum essentialoilwasobtainedfromthejuiceindustrypeel residue, which was pressed and the oil was washed out with water of the material, while the dried fruit of P. nigrum was steam distilled for 60 minutes to obtain the essential oil.
2.2. Analysis by GC/MS. The crude essential oil was analyzed on a GC-MS QP2010 (SHIMADZU) gas chromatograph equipped with a mass spectrometer using electron impact ionization detection. The gas chromatographic (GC) conditions were as follows: injector temperature: 260[degrees]C; detector temperature: 290[degrees]C; carrier gas (Helium): flow rate 1 mL/min; and split injection with split ratio 1 : 40. Oven temperature was raised from 60[degrees]C to 290[degrees]C at a rate of 3[degrees]C/min. One microliter of each sample, dissolved in CH2Cl2 (1: 100 mg/[micro]L), was injected on a RTX-5 column (i.d. = 0.25 mm, length 30 m, film thickness = 0.25 [micro]m). Mass spectra (MS) were recorded 70 eV and scan rate of 1 scan/s. The retention indices were calculated by interpolation of retention times of the substances to the retention times of a mixture of aliphatic hydrocarbons (C7-C40) (Sigma-Aldrich Corporation, St Louis, MO) analyzed in the same conditions (Van den Dool H. & Kratz). The identification of substances was performed by comparison of their retention indices and mass spectra with those reported in literature . The MS fragmentation pattern of compounds was also checked with NIST mass spectra libraries.
2.3. Preparation of the Samples for Acaricidal Test. The experiment was conducted in two phases. Essential oils were tested separately and after mixing were performed at equal concentrations, in 1 : 1 proportions (50% Citrus limonum and 50% Piper nigrum). The essential oils of C. limonum and P. nigrum were dissolved in an aqueous solution with 2% dimethyl sulfoxide (v/v) as an emulsifying agent, as described by Gazim et al. in . Solutions were serially diluted in order to obtain the concentrations of 2.5%, 5%, and 10% (v/v), obtaining a final volume of 5mL. The emulsifying solution (2% DMSO) was used as the negative control. A control with water was also carried out. Amitraz (2 [micro]L/mL) and Deltamethrin (1 [micro]L/mL) in water were used as positive control. The test was repeated three times in duplicate.
2.4. Rhipicephalus microplus Ticks for Bioassays. The engorged females ticks of R. (B.) microplus were collected from infested animals from a farm of Rio Grande do Sul (Brazil) without history of acaricide use. The tick population was maintained based on artificial infestations on calves at Faculdade de Veterirnria from Federal University of Rio Grande do Sul (UFRS), Brazil. All experiments were conducted following the guidelines of the Ethics Committee on Animal Experimentation of UFRS and FEPAGRO. The ticks were thoroughly washed with tap water, dried on filter paper towel, and used in the adult immersion test.
2.5. Adult Immersion Test (AIT). The AIT was performed as described by Drummond et al.  with minor modifications. The ticks were distributed to groups randomly (20 engorged females per group). The different groups of R. (B.) microplus were immersed for 1min in 5mL of the respective concentrations (2.5%, 5%, and 10%) of C. limonum and P. nigrum by placing them directly into a 50 mL Falcontype tube which was gently agitated at room temperature. In experiments of additive effects we used the ratio 1 : 1 (vol/vol) of each oil, with a final concentration of 2.5%, 5%, and 10%. Ticks were recovered from the solutions and dried and each group was transferred to Petri dish (9 cm diameter, 1,5 cm high), weighed, and kept in BOD incubator at a temperature of 28[degrees]C and relative humidity of 70-80% for oviposition. This experiment was performed three times in duplicate. The emulsifying solution (2% DMSO) was used as the negative control, while Amitraz (2 [micro]L/mL) and Deltamethrin (1 [micro]L/mL) in water were used as positive control. The mortality was observed during 15 days. The dead ticks were diagnosed based on three specific signs and features: increasing cuticle darkness and stopping Malpighian tube movement and hemorrhagic skin lesions. After 15 days, the eggs laid were placed in glass tube, weighed, and observed separately at the same condition of incubation for the next 30 days for visual estimation of hatching rate.
The percentage inhibition of oviposition was calculated as follows.
Reproductive index (RI) = average weight of eggs laid (mg)/average weight of females before treatment (g).
Percentage inhibition of oviposition (IO%) = RI of control group--RI of treated group/RI control group x 100.
2.6. Statistical Analyses. Data were expressed as the mean [+ or -] standard error of the mean (SEM). Groups were compared using ANOVA one-way test. A p value of less than 0.05 was considered significant. Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, USA) software.
The efficacy was assessed by measuring female mortality (%) and the lethal concentrations for 50% (LC50) and 90% (LC90) with their 95% confidence limits (CL) values were estimated by applying regression equation analysis to the probit transformed data of mortality.
3.1. Identification of the Components by GC/MS. The composition of the oil from Citrus limonum and Piper nigrum, together with the relative retention index and percentage of the identified compounds, is listed in Table 1. A total of 29 compounds representing 96.3% of the total oil were identified from C. limonum essential oil. The chromatographic profile of C. limonum showed a complex mixture of monoterpenes (90.3%) and sesquiterpenes (3.1%). C. limonum oil was composed mainly by the monoterpenes limonene (50.3%), [beta]-pinene (14.4%), and y-terpinene (11.7%).
In P. nigrum essential oil, 42 compounds were identified, comprising 89% of the oil. The essential oil was characterized by the presence of sesquiterpenes (58.9%) and monoterpenes (26.3%). The sesquiterpene [beta]-caryophyllene was identified as the major compound, representing 26.2% of the oil, followed by the monoterpenes hydrocarbons u-ocymene (5.8%) and a-pinene (5.5%).
3.2. Rhipicephalus (Boophilus) microplus Engorged Female Mortality Rate. Laboratory tests were carried out to determine the toxicity of the essential oils from C. limonum and P. nigrum on engorged females of R. (B.) microplus. The results referents to mortality rate of engorged females after adult immersion test (AIT) are present in Figure 1 and Table 2. Essential oil from C. limonum exhibited 90% of mortality on engorged females in the highest concentration tested after 48 h, reaching 100% at the end of the experiments (day 16). At concentration of 5%, C. limonum achieved approximately 80% mortality rate in the fourth day and getting the most for 90% mortality at the 12th day. The 2.5% C. limonum essential oil led to 50% mortality rate on day 16 after treatment (Figure 1(a)).
The essential oil of P. nigrum also demonstrated high efficacy against R. (B.) microplus tick using the highest concentration (10%), which caused a mortality of 80% of females on the 16th day. The concentrations of 5 and 2.5% essential oil led to a mortality rate of 60% and 35%, respectively (Figure 1(b))
The toxic effect of the mixture of essential oils was also studied (Figure 1(c)). The mixture of C. limonum and P. nigrum oil induced 100% death at a concentration of 10% at the end of treatments. The results of the groups treated with the combination of the oils, at all concentrations tested, were very similar to the results of the C. limonum oil. These results demonstrate the dose-dependent character of R. (B.) microplus mortality and there was a significant difference between all treatments when compared with control group.
When AIT was performed with positive controls, the mortality of engorged females of R. (B.) microplus for Deltamethrin and Amitraz was 57.7% and 27.7%, showing a level of resistance status (Figure 1(d))
Table 2 summarizes LC50 and LC90 values for individuals and combined treatments of C. limonum and P. nigrum oils against engorged females. The most potent modified oil was C. limonum with a LC90 of 4.9% whereas the combined oils exhibited a LC90 of 5.1%.
3.3. Effect of Essential Oil of Citrus limonum, Piper nigrum, and Their Mixture on Rhipicephalus (Boophilus) microplus Female Oviposition and Hatching. In the AIT the efficacy of treatment against engorged females was also evaluated by measuring egg production. The results are presented in Table 3. Ours results showed that C. limonum and the oil mixture have the best inhibition of oviposition rate, which is 100% in a dilution of 10%. However, at 5% the group with combined oils exhibited greater inhibition of oviposition with 94%, compared to 81.6% inhibition by individual C. limonum. P. nigrum essential oil, at concentrations of 10%, 5%, and 2.5%, inhibited egg-laying by 96%, 83%, and 50%, respectively. Data were significantly different in relation to the control (DMSO 2% and water) group (p < 0.001). The few eggs laid by the females treated with 5% of C. limonum essential oil have not hatched into larvae. This result showed that oviposition is also dose-dependent (Table 3). Thus, analysis of variance p < 0.05 for treatments confirmed the positive development of inhibition oviposition with increasing concentration. The mixture is effective against R. (B.) microplus engorged females since its effect is fast and is expressed at a low concentration on reducing the rate of lay. However, the essential oils did not interfere in the hatchability of the larvae.
The analysis of chemical composition of Citrus limonum essential oil by GC/MS showed as major compounds limonene (50.3%), [beta]-pinene(14.4%), and [gamma]-terpinene (11.7%). This result agrees with that found by others groups in previous studies with species of genus Citrus, where the same chemical compounds can be found, although in different proportions . The essential oil chemical compositions of Piper nigrum consisted primarily of the sesquiterpene [beta]-caryophyllene and the monoterpenes hydrocarbons <rocymene and a-pinene, at 26.2%, 5.8%, and 5.5%, respectively.
Essential oil from C. limonum exhibited greater activity on engorged females than P. nigrum, reaching a mortality of 100% at concentrations of 10%, on 16th day. The values of LC50 and LC90 of C. limonum were 2.2% and 4,9%, respectively. P. nigrum exhibited LC50 of 3.7% and LC90 of 14.8%.
Components such as limonene and pinene have already been reported to possess acaricidal activity against ticks. Limonene, extracted from Lippia alba (Verbenaceae) essential oils of two chemotypes, demonstrated toxic effect on R. (B.) microplus larvae . In another study, it was shown that limonene inhibited 91% of R. (B.) microplus hatching eggs at concentration of 1.25 [micro]g/mL . Moreover, this chemical constituent has also exhibited insecticide potential and proved to be highly toxic to mosquito larvae of Aedes aegypti (Diptera: Culicidae), Culex pipiens pallens (Diptera: Culicidae), and Ochlerotatus togoi (Diptera: Culicidae) , for grain insect pests stored Rhyzopertha dominica (Coleoptera: Bostrichidae) and Tribolium castaneum (Coleoptera: Tenebrionidae)  and lice Pediculus humanus capitis (Anoplura: Pediculidae) . Acaricide activity in R. (B.) microplus larvae has also been demonstrated with essential oil of Cunila incana (Lamiaceae), rich in [beta]-pinene (27.5%) and [alpine]-pinene (26.7%), reaching 100% mortality with concentration of 2.5 [micro]L/mL . This suggests the major constituents of the essential oil are acting in an additive or added effect manner, thus contributing equally to the observed toxic effects. However, the added effect action of other minor constituents cannot be disregarded. Some studies have shown that essential oils have higher efficiency than their isolated components [33, 34].
Chungsamarnyart and Jansawan in  observed that the essential oil of Citrus maxima and Citrus sinensis showed over 90% mortality of R. (B.) microplus engorged females at a concentration of 1/5 in 24 hours. This concentration is higher than that found in our study. Already Citrus reticulata oil demonstrated a similar profile to C. limonum, reaching about 95% mortality within 24 h at a concentration of 1/10. Plants of the same genus could have the same class of substances and so present similar activity. However, such substances may vary in concentration according to seasonality, circadian rhythm, and development of the plant .
Citrus limonum has already shown biological activity in several species of insects. The essential oil of C. limonum caused antifeedant activity in Spodoptera litura larvae (Lepidoptera: Noctuidae) and took the mortality Spodoptera littoralis larvae (Lepidoptera: Noctuidae) in the test by contact [37, 38]. Pavela in  also demonstrated that essential oil of these specie showed fumigant and contact activity on the house fly Musca domestica (Diptera: Muscidae).
The reduction in the number of C. limonum eggs is probably due to female mortality in the first few days after treatment. However, it is possible that the essential oil of P. nigrum has caused a reduction in female's fecundity, since exposure to sublethal concentrations of the oil significantly reduced the number of eggs. The same effect was observed with the essential oil of Piper marginatum used as fumigant against the mite Tetranychus urticae (Acari: Tetranychidae), where the fecunditywas drastically reduced. The [beta]-caryophyllene was one of the main compounds (16%) found in the essential oil of P marginatum, as well as the oil of P. nigrum in the present work. This constituent was also evaluated against T.urticae and this also reduced the females' fecundity in sublethal concentrations .
According to chemical analysis, the essential oil of P. nigrum majority has the sesquiterpene [beta]-caryophyllene (26.2%) and the monoterpenes hydrocarbons [sigma]-ocymene (5.8%) and [alpha]-pinene (5.5%). However, Ferreira et al. in  analyzed the essential oil from P. nigrum and found as main components the sesquiterpene [beta]-caryophyllene (21.8%) and the monoterpenes limonene (19.8%) and 3-[sigma]-carene (14.3%). The constituents of essential oils can vary with environmental conditions, such as climate, soil type and brightness. The biological properties of [beta]-caryophyllene have been confirmed in previous studies involving Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), S. littoralis , larvae of A. aegypti , and T. urticae . The compound [alpha]-pinene has been reported with insecticidal activity in larvae of C. pipiens  and as a fumigant against the adult mushroom fly Lycoriella mali (Diptera: Sciaridae) .
Plants of the genus Piper have demonstrated toxic effect on R. (B.) microplus. Ferraz et al. in  showed that the essential oils of Piper mikanianum and Piper xylosteoides were active against R. (B.) microplus larvae (LC50 of 2.3 and 6.2 [micro]L/mL, resp.). Piper tuberculatum and Piper aduncum extracts were also highly effective against the larvae and engorged female tick R. (B.) microplus [48, 49]. In addition, other studies have shown that the essential oil of P nigrum hasinsecticidal activityagainstmosquitoes A. aegypti, A. stephensi (Diptera: Culicidae), and C. quinquefasciatus (Diptera: Culicidae) .
Ours results showed that the oil mixture has a high acaricidal activity on engorged females of R. (B.) microplus, reaching 100% mortality, at a concentration a little larger than that of the C. limonum essential oil (LC90 = 4,9%). However, the oils combination showed to be more effective in the inhibition of oviposition compared to the oil C. limonum. Considering that C. limonum oil caused high mortality of females and P. nigrum oil reduced fertility, it is possible that the essential oils have an additive effect when mixed in the same solution, exhibiting both effects against R (B.) microplus tick females. Yessinou et al. in  recently showed that the mixture of Syzygium aromaticum (Myrtaceae) and Cymbopogon citratus (Poaceae) essential oils was more effective in engorged females of R. (B.) microplus than when tested separately, confirming the action potential of essential oils when used simultaneously.
This study demonstrates that the essential oil of lemon has a high acaricide activity against the tick R. (B.) microplus, especially in its engorged female and egg stages, cutting its cycle. Thus, it can be a possible candidate for biocontrol against this cattle tick.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
The authors are grateful to FAPERJ, CNPq FOPESQ-UFF for financial support to the present work.
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Rafaelle Vinturelle, (1,2) Camila Mattos, (1,2) Jessica Meloni, (1,2) Jeane Nogueira, (3,4) Maria Julia Nunes, (5,6) Itabajara S. Vaz Jr., (7,8) Leandro Rocha, (3,4) Viviane Lione, (9) Helena C. Castro, (2) Evelize Folly das Chagas (1,2,8)
(1) Laboratorio deEstudos em Pragas eParasitos, Universidade Federal Fluminense, IB, Departamento de Biologia Celular e Molecular-GCM, Niterai, RJ, Brazil
(2) Programa de Pos-Graduacao em Ciencias e Biotecnologia, Universidade Federal Fluminense, Niteroi, RJ, Brazil
(3) Laboratario de Tecnologia de Produtos Naturais, Faculdade de Farmacia, Universidade Federal Fluminense, Niteroi, RJ, Brazil
(4) Programa de Pos-Graduacao em Ciencias Aplicadas a Produtos para Saude, Departamento de Farmacia e Administracao Farmaceutica, Faculdade de Farmacia, Universidade Federal Fluminense, Rua Doutor Mario Viana 523, 24241-000 Niteroi, RJ, Brazil
(5) Bellarome Aromoterapia, Centro, Rio de Janeiro, RJ, Brazil
(6) Centro Universitario Augusto Motta (UNISUAM), Rio de Janeiro, RJ, Brazil
(7) Centro de Biotecnologia e Faculdade de Veterinaria, Universidade de Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
(8) Instituto Nacional de Ciencias e Tecnologia em Entomologia Molecular (INCT-EM), Rio de Janeiro, RJ, Brazil
(9) Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
Correspondence should be addressed to Evelize Folly das Chagas; email@example.com
Received 30 March 2017; Accepted 25 July 2017; Published 8 October 2017
Academic Editor: Jan A. Miernyk
Caption: Figure 1: Mortality percentage of R. microplus engorged females exposed to Citrus limonum and Piper nigrum essential oil. Adult Immersion Test (AIT) on mortality of the tick R. microplus. Groups treated with different concentrations of essential oil of C. limonum (a), P nigrum (b), the combination of both oils (c), and positive control were the commercial acaricides Deltamethrin and Amitraz. In combination oils used the 1: 1 (vol/vol) concentration of each oil. Results are means [+ or -] SD of five experiments.
Table 1: Percentage composition of Citrus limonum and Piper nigrum oils. Constituents RI Citrus Piper limonum (%) nigrum (%) Monoterpenes hydrocarbons [alpha]-Thujene 927 0.6 1.0 [alpha]-Pinene 935 2.6 5.5 Camphene 950 0.1 -- Sabinene 975 2.8 0.9 [beta]-Pinene 978 14.4 4.1 Myrcene 992 2.0 0.3 Phellandrene 1007 -- 4.1 [delta]-3-Carene 1013 -- 0.9 [alpha]-Terpinene 1018 0.2 0.7 [sigma]-Ocymene 1026 2.9 5.8 Limonene 1030 50.3 2.1 [beta]-E-Ocymene 1037 0.1 -- [beta]-Z-Ocymene 1048 0.1 -- [gamma]-Terpinene 1060 11.8 0.5 Terpinolene 1086 0.1 -- p-Menth[alpha]-2,4(8)-Diene 1091 -- 0.3 Oxygenated monoterpenes 1,8-Cineol 1034 0.1 -- Linalool 1101 0.2 -- Limonene oxide -cis 1135 0.1 -- Limonene oxide -trans 1139 0.1 -- Carvenon 1147 0.1 -- Terpinen-4-ol 1178 0.1 -- [alpha]-Terpineol 1191 0.4 0.1 Nerol 1224 0.1 -- Neral 1242 1.1 -- Sesquiterpene hydrocarbons [delta]-Elemene 1340 -- 0.9 [alpha]-Cubebene 1353 -- 0.5 [alpha]-Copaene 1379 -- 4.2 [beta]-Cubebene 1383 -- 0.2 [beta]-Elemene 1395 -- 1.1 Cyperene 1404 -- 0.2 Bergamotene 1418 0.04 -- [beta]-Caryophyllene 1426 -- 26.2 [beta]-Copaene 1429 -- 0.2 Trans- Bergam otene 1439 -- 3.9 [alpha]-Germanene 1443 -- 0.5 (z)-[beta]-Farnese 1446 -- 0.2 [alpha]-Humulene 1458 -- 2.9 9-EPI-(E)-Caryophyllene 1466 -- 0.3 [gamma]-Muurolene 1481 -- 0.9 Trans-Muurol[alpha]-4(14), 1485 -- 1.5 5-Diene [beta]-Selinene 1491 -- 1.1 [alpha]-Selinene 1499 -- 1.1 Bisabolene 1506 0.1 -- [beta]-Bisabolene 1512 1.8 4.1 [gamma]-Cadinene 1519 -- 0.6 [delta]-Cadinene 1528 -- 2.1 Germacrene B 1562 -- 0.5 Oxygenated sesquiterpenes Bergamotol [alpha]-trans 1439 1.1 -- Caryophyllene oxide 1589 -- 4.2 Junenol 1624 -- 0.6 Epi-[alpha]-Muurolol 1648 -- 0.4 [alpha]-Muurolol 1652 -- 0.3 [alpha]-Cadinol 1660 -- 0.4 Others Neril format 1272 1.9 -- Neril acetate 1367 0.9 -- Geranyl acetate 1386 0.3 -- Guaiol acetate 1729 -- 0.7 Benzil benzoato 1771 -- 3.0 Manool 2063 -- 0.2 Total identified 96.3 89.0 RI: retention index on DB-5MS column in reference to n-alkanes. Table 2: LC50 and LC90 (%) obtained for the engorged female of the essential oils of Citrus limonum, Piper nigrum, and their combination on R. microplus. Essential oil LC50 (%) LC90 (%) Citrus limonum 2.2 4.9 Piper nigrum 3.7 14.8 C. limonum + P. nigrum 2.2 5.1 Table 3: Mortality percentage (MAM) of R. microplus engorged females, exposed to different concentrations of Citrus limonum and Piper nigrum, and effect reproductive index (RI), inhibition of oviposition (IO), and larvae hatching (LH). Concentration (%) MAM (%) [+ or -] SE RI [+ or -] SE Citrus limonum 10% 100.0 [+ or -] 0.0 (a, b) 0.0 [+ or -] 0.0 (a, b) 5% 94.4 [+ or -] 5.7 (a, b) 0.1 [+ or -] 0.1 (a, b) 2.5% 52.8 [+ or -] 12.8 (a, b) 0.3 [+ or -] 0.1 (a, b) Piper nigrum 10% 81.7 [+ or -] 6.7 (a, b) 0.0 [+ or -] 0.0 (a, b) 5% 59.4 [+ or -] 9.4 (a, b) 0.1 [+ or -] 0.0 (a, b) 2.5% 36.1 [+ or -] 3.9a 0.2 [+ or -] 0.0 (a, b) C. limonum + P. nigrum 10% 100.0 [+ or -] 0.0 (a, b) 0.0 [+ or -] 0.0 (a, b) 5% 90.0 [+ or -] 10.0 (a, b) 0.0 [+ or -] 0.0 (a, b) 2.5% 55.6 [+ or -] 9.0 (a, b) 0.1 [+ or -] 0.0 (a, b) Amitraz (2 278 [+ or -] 7.9a 0.0ab [micro]L/mL) Deltamethrin 578 [+ or -] 9.0 (a, b) 0.0ab (1 [micro]L/mL) DMSO 2% 16.7 [+ or -] 3.4 0.5 [+ or -] 0.0 Water 3.3 [+ or -] 3.3 0.5 [+ or -] 0.0 Concentration (%) IO (%) LH Citrus limonum 10% 100 -- 5% 81.6 Without hatching 2.5% 35 There was hatching Piper nigrum 10% 96 Without hatching 5% 83 There was hatching 2.5% 50 There was hatching C. limonum + P. nigrum 10% 100 -- 5% 94 Without hatching 2.5% 67 There was hatching Amitraz (2 100 -- [micro]L/mL) Deltamethrin 100 -- (1 [micro]L/mL) DMSO 2% 0 There was hatching Water 0 There was hatching MAM: mean % adult mortality within 16 days, SE: standard error, RI: reproductive index, IO: (%) percent inhibition of oviposition, and LH: Larvae hatching after females treatment. (a) Significant difference in relation to the negative control (water). (b) Significant difference in relation to the negative control (2% DMSO) (ANOVA one way p =< 0.0001).
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|Title Annotation:||Research Article|
|Author:||Vinturelle, Rafaelle; Mattos, Camila; Meloni, Jessica; Nogueira, Jeane; Julia Nunes, Maria; Vaz, Ita|
|Publication:||Biochemistry Research International|
|Date:||Jan 1, 2017|
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