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AMAZONICA http://dx.doi.org/10.1590/1809-4392201700053

ORIGINAL ARTICLE

Acaricidal activity of extracts from different structures

of Piper tuberculatum against larvae and adults of
Rhipicephalus microplus
Andrina Guimarães Silva BRAGA1, Krishna Ferri Alencar de SOUZA2, Fabio da Silva BARBIERI3*,
Cleberson de Freitas FERNANDES4, Rodrigo Barros ROCHA2, José Roberto VIEIRA JUNIOR2,
Clariana Lins LACERDA2, Caroline Oliveira CELESTINO2, Valdir Alves FACUNDO5,
Luciana Gatto BRITO3
1
Universidade Federal do Amazonas, Programa de Pós-Graduação em Biodiversidade e Biotecnologia (BioNorte), Setor Sul, Bloco M, Av. Gal. Rodrigo Otávio Jordão
Ramos 3000, CEP 69077-000 Manaus, Amazonas, Brasil
2
Embrapa Rondônia, Rodovia BR-364, Km 5,5, Zona Rural, Caixa Postal 127, CEP 76815-800 Porto Velho, Rondônia, Brasil
3
Embrapa Amazônia Oriental, Trav. Dr. Enéas Pinheiro, s/n, Bairro Marco, Caixa postal 48, CEP 66095-903 Belém, Pará, Brasil
4
Embrapa Agroindústria Tropical, Rua Dra. Sara Mesquita 2270, Bairro Planalto do Pici, CEP 60511-110 Fortaleza, Ceará, Brasil
5
Universidade Federal de Rondônia, BR-364, Km 9,5, CEP 76801-059 Porto Velho, Rondônia, Brasil
* Corresponding author: fabio.barbieri@embrapa.br

ABSTRACT
The strategies to control the cattle tick, Rhipicephalus microplus are based mainly on the use of synthetic pesticides. However,
the emergence, establishment, and development of resistance of ticks is rendering the main chemical groups ineffective.
Finding new molecules to effectively control infestations by R. microplus is necessary to maintain the productivity of cattle
herds, particularly of taurine breeds established in equatorial and tropical regions of the world. Ethanol extracts from the
leaves, stems, and fruits of Piper tuberculatum were evaluated in bioassays at concentrations of 50, 25, 12.50, 6.25, 3.12 and
1.56 mg mL-1. The concentrations lethal to 50% of the individuals (LC50) of tick larvae after 24 hours of exposure were 3.62,
3.99 and 5.30 mg mL-1 for fruit, stem and leaf extracts, respectively. Against the engorged females, the highest efficacy rates
were obtained at the concentration of 50 mg mL-1, corresponding to 71.57%, 68.38% and 37.03% of the fruit, leaf and stem
extracts, respectively. The main effect of the ethanol extracts was on the egg hatching rate of ticks, with a reduction of 55.63%
for the fruit and leaf extracts, and 20.82% for the stem extract. The results show that P. tuberculatum is a promising source of
molecules for use as active ingredients in pesticide formulations for R. microplus control.
KEYWORDS: cattle tick, bioassay, active molecules, Piperaceae

Atividade acaricida de extratos de diferentes estruturas de

Piper tuberculatum sobre larvas e adultos de Rhipicephalus microplus
RESUMO
Estratégias de controle do carrapato dos bovinos, Rhipicephalus microplus, fundamentam-se na utilização de pesticidas. Os
principais grupos químicos utilizados atualmente mostram-se ineficazes devido ao surgimento de populações resistentes. A
pesquisa de novas moléculas com eficiência acaricida é uma necessidade para manutenção da produtividade dos rebanhos bovinos
estabelecidos em regiões de clima tropical. Avaliamos a atividade de Piper tuberculatum para o controle de fêmeas ingurgitadas
e larvas de R. microplus através de bioensaios de imersão de adultos e de pacotes de larvas. As concentrações avaliadas foram
de 50; 25; 12,50; 6,25; 3,12 e 1,56 mg mL-1 de extrato etanólico de folha, talo e fruto de P. tuberculatum. As concentrações
letais para 50% dos indivíduos (CL50) após 24 horas de exposição de larvas de R. microplus foram 3,62; 3,99 e 5,30 mg mL-1
para os extratos etanólicos de fruto, talo e folha, respectivamente. Para fêmeas ingurgitadas, a maior eficácia resultou da
concentração de 50 mg mL-1 de extrato de fruto (71.57%). O principal efeito dos extratos etanólicos de P. tuberculatum foi
sobre a eclodibilidade, com uma redução de 55.63% para extratos de fruto e folha. P. tuberculatum mostra-se promissora como
fonte de moléculas candidatas a uso em pesticidas, em formulações destinadas ao controle das infestações de R. microplus.
PALAVRAS-CHAVE: carrapato dos bovinos, bioensaios, biomoléculas ativas, Piperaceae

CITE AS: Braga, A.G.S.; Souza, K.F.A.; Barbieri, F.S.; Fernandes, C.F.; Rocha, R.B.; Vieira Junior, J.R.; Lacerda, C.L.; Celestino, C.O.; Facundo, V.A.; Brito,
L.G. 2018. Acaricidal activity of extracts from different structures of Piper tuberculatum against larvae and adults of Rhipicephalus microplus. Acta
Amazonica 48: 57-62.

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INTRODUCTION MATERIAL AND METHODS

The global economic losses to cattle breeders caused by pests Collection of the plant material
due to lower milk and meat production, hide damage and
Leaves, stems, and fruits of Piper tuberculatum were collected
treatment costs are enormous. The latest data indicate that
in the native forest reserve area of the Federal University of
in Brazil alone, these losses amount to US$ 6 billion a year,
Rondônia (63°56’22”W, 8°50’03”S), in the municipality of
of which 50% are attributed to the cattle tick, Rhipicephalus
Porto Velho, state of Rondônia (southwestern Amazon) in
microplus (Canes.) (Grisi et al. 2014).
January 2013. Prepared botanical vouchers were sent to the
The primary method used to minimize these losses is the Herbarium of the National Research Institute of the Amazon
application of synthetic pesticides. However, the incorrect (INPA) for identification confirmation, and registered under
and indiscriminate use of these products has caused the number 211724.
development of cattle tick populations that are resistant to the
various commercially available chemical groups (Brito et al. Extract preparation and dilution
2011). The search for new molecules with acaricidal activity Leaves (1.40 kg), stems (1.60 kg) and fruits (0.30 kg) were
is currently one of the greatest challenges to maintaining the submitted to extraction with ethanol PA (3 L) for seven days,
sustainability of cattle breeding in tropical regions. to obtain extracts from leaves (PTLEt, 87 g), stems (PTSEt, 36
The promising biocidal activity of various plants to control g) and fruits (PTFEt, 46.90 g). For the bioassays, the solutions
different pathogenic vectors and agents that impair livestock of the three ethanol extracts of P. tuberculatum were diluted in
and human health has been demonstrated by various studies ethanol (larval packet test) and water + Tween 20 at 2% (adult
(Klauck et al. 2014; Custódio et al. 2016; Rodrigues et al. immersion test). The gross extracts were weighed and diluted
2017; Fatemi et al. 2017). The active research interest in the in the solvents with the aid of ultrasound and a vortex agitator
chemical components produced by the metabolism of plants to maximize the solubilization. The final concentrations of 50,
has led to the isolation of various substances that take part in 25, 12.50, 6.25, 3.12 and 1.56 mg mL-1 were obtained after
the defense mechanisms of plants against attack by pests and dissolving the crude extract. The extracts at a concentration
diseases. Among these substances, alkaloids, steroids, terpenes, higher than 50 mg mL-1 had high density and viscosity, so
phenylpropanoids, lignans, flavonoids, and amides stand out they were not evaluated.
as promising bioactive molecules from plants for use in the Tick preparation and bioassays
health sciences (Parmar et al. 1997).
Engorged females were collected from naturally infested
Approximately 55 plant species have been evaluated cattle maintained at the Experimental Field of Porto Velho of
regarding their potential use to control R. microplus Embrapa Rondônia (63°50’58”W, 8°48’10”S). Immediately
(Borges et al. 2011),. Among these, Piper tuberculatum after collection, the ticks were immersed in a 2% sodium
Jacq. (Piperaceae), popularly known in Brazil as “pimenta hypochlorite solution, dried on paper towels and selected
d’arda” or “pimenta longa”, stands out as a promising according to integrity, motility, and degree of engorgement.
species that produces bioactive molecules with potential use
The larvae used in the larval packet test (LPT) came from
to control pests and diseases that affect both crops (Scott
30 engorged females. These ticks were attached dorsally with
et al. 2002; Castro et al. 2008; Trindade et al. 2012) and
two-sided tape in Petri dishes and placed in a BOD chamber
livestock (Chagas et al. 2012; Lima et al. 2014). Extracts of
at a temperature of 27 +/- 1º C and relative humidity above
P. tuberculatum also have proven action against protozoa of
80% for egg laying. After 18 days of oviposition, the egg
medical importance such as Leishmania amazonensis (Ferreira
masses were removed and placed in plastic tubes plugged
et al. 2010) and Trypanossoma cruzi (Regasini et al. 2009). It
with hydrophilic cotton and kept in the BOD chamber under
also has molluscicidal effect against Biomphalaria glabrata,
the same temperature and humidity conditions previously
the intermediate host of Schistosoma mansoni (Rapado et al.
described, until hatching of the larvae. Bioassays were
2011; Rapado et al. 2013).
conducted in triplicate for each concentration of the extracts,
This study involved the testing of ethanol extracts of the both in larval and engorged female tests.
leaves, stems, and fruits of P. tuberculatum for control of R.
microplus, to determine what structures of this plant have Larvae sensitivity on impregnated paper
stronger acaricidal activity and can thus be used as a source The bioassays to assess the efficiency of the extract on R.
to synthesize new active ingredients and a bioactive product microplus larvae were performed according to Stone and
for control of cattle tick populations. The evaluations were Haydock (1962), as modified by Miller et al. (2002). The
performed through the bioassays: 1) Adult Immersion Test packets were made of filter paper and impregnated with each
(TIA) for engorged females; and 2) Larval Packet Test (LPT) concentration of the stem, leaf and fruit extracts diluted in
for the larval stage. alcohol. As controls, packets were impregnated with ethanol

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(negative control) and technical grade cypermethrin diluted in Statistical analysis

acetone at a concentration of 25.60 µg mL-1 (positive control). The 50% lethal concentration (LC50) was calculated for larvae
Approximately 100 larvae were placed in each packet, using packet test mortality data in regression analysis using
which was immediately sealed with paper clips and put in the the Probit test, performed with the BioStat 2009 Professional
BOD chamber at a temperature of 27 +/- 1º C and humidity 5.8.4 software. Variables from the adult immersion test
greater than 80%, where they remained for 2 hours. After this were analyzed using ANOVA with a factorial design of 6x3
interval, the packets were opened, and the living and dead (concentration x extracts) followed by the Scott and Knott’s
larvae were counted in each replicate to calculate mortality. test at 5% significance level using the GENES Program
(Cruz, 2016).
Immersion test with engorged females
The acaricidal potential of the stem, leaf and fruit extracts
of P. tuberculatum on engorged females was analyzed by the
RESULTS
adult immersion test (AIT), as described by Drummond et The leaf and stem extracts caused 100% mortality of the
al. (1973). Each replicate consisted of a group of ten females, larvae starting at the concentration of 12.50 mg mL-1, while
with homogeneous weight, immersed for five minutes in 10 ml the fruit extract achieved maximum mortality of 96.20% only
of extract at each of the concentrations evaluated. As controls at the concentration of 50 mg mL-1 (Figure 1). Although the
we used technical grade cypermethrin diluted in acetone at a fruit extract did not produce 100% mortality, at the lowest
concentration of 25.60 µg mL-1 (positive control) and water concentrations it performed better than the leaf and stem
+ Tween 20 at 2% (negative control). After immersion, the extracts. The fruit extract also had the lowest LC50 value, 3.62
engorged females were dried on a paper towel and mounted ± 2.21 (0.73 - 17.98) mg mL-1 (Table 1). The mortality of
dorsally in Petri dishes with two-sided tape. The plates were the negative control was below 5% and of the positive control
kept in the BOD chamber at a temperature of 27 +/- 1º C above 90% demonstrating the quality of the test.
and humidity above 80%. No significant differences of percentage reduction of
After the end of oviposition, the egg masses of each group oviposition were observed in relation to the concentration
were weighed and allocated in labeled plastic syringes plugged among the three extracts. A significant difference was only
with cotton and incubated in the BOD under the same observed for the average percentage reduction of oviposition
temperature and humidity conditions as described previously caused by the PTLEt at concentrations of 3.12 e 1.56 mg
for larval hatching. The number of hatched and not hatched mL-1 when compared to the other concentrations (Table 2).
eggs were counted after 16 days to determine the percentage With respect to the percentage reduction of hatching,
reduction of oviposition (% OR) and hatching (% HR) the comparison of the averages for the extracts PTLEt,
(Gonzales 2003). Estimated reproduction (ER), and treatment PTSEt and PTFEt revealed a significant difference among
efficacy (E) were calculated according to Drummond et al. the concentrations of 1.56, 6.25 and 12.50 mg mL-1 for
(1973) as follows: the three extracts, where the highest averages for these

Figure 1. Mortality of Rhipicephalus microplus larvae in ethanol extracts of the leaf (PTLEt), stem (PTSEt) and fruit (PTFEt) of Piper tuberculatum in the larval packet test.
Negative control is represented by C (-) and positive control by C (+).

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concentrations were observed for PTFEt. For all the extracts, The average estimated reproduction measures declined
the highest percentages reduction of hatching occurred at the significantly for the engorged females immersed in PTLEt and
concentration of 50 mg mL-1. For PTFEt, the concentrations PTSEt. For all the extracts, the concentration of 50 mg mL-1
of 6.25 and 12.50 mg mL-1 caused the same reduction (the was most effective in reducing the estimated reproduction
greatest hatching reduction for this extract) (Table 2). (Table 2). The concentration of 50 mg mL-1 of both the PTFEt
and PTLEt extracts presented the best performance in the
AIT, with treatment efficacy levels of 71.57% and 68.38%,
Table 1. LC50 (50% lethal concentration) of the ethanol extracts of the leaf (PTLEt),
stem (PTSEt) and fruit (PTFEt) of Piper tuberculatum on larvae of Rhipicephalus
respectively (Table 2). There were no viable postures in the
microplus. Values are means of three replicates followed by the standard deviation female groups of the positive control, demonstrating high
and the range. pyrethroid efficacy in the evaluated population of R. microplus.
Extract LC50(mg mL-1)
PTLEt 5.30 ± 0.84 (3.42 - 8.21)
DISCUSSION
Leaf extracts of P. tuberculatum have previously been studied
PTSEt 3.99 ± 0.80 (2.18 - 6.60)
as insecticides (Trindade et al. 2012; Castro et al. 2008),
PTFEt 3.62± 2.21 (0.73 – 17.98)
however, very few studies have been conducted to assess the use
of this plant’s extracts against ticks (Chagas et al. 2012). The
efficiency of an acaricide can vary according to the extraction
Table 2. Average percentage reduction oviposition (%OR), hatching reduction
percentages (%HR), estimated reproduction (ER), and efficacy (E) in the adult method, solvent polarity and plant part used to obtain the
immersion test (AIT) with ethanol extracts of leaf (PTLEt), stem (PTSEt) and fruit extract (Lima et al. 2014).
(PTFEt) of Piper tuberculatum on engorged Rhipicephalus microplus females. All three extracts showed promise against the larvae of
Concentration (Conc.) in mg mL-1. Numbers are means followed by the standard
deviation. Equal upper-case letters in the row and lower-case letters in the column
Rhipicephalus microplus. Chagas et al. (2012) observed similar
indicate no difference by the Scott-Knott test at 5% significance level. results (LC50=0.41%≈4.10 mg mL-1) using leaf extract of P.
tuberculatum to control R. microplus larvae. Lima et al. (2014)
Conc. PTLEt PTSEt PTFEt obtained a LC50 value approximately 50 times lower than that
50 35.01±16.20Aa 26.04±4.98Aa 31.91±7.03Aa observed in this study for the ethanol extracts from the fruit
25 30.38±3.71Aa 27.33±7.09Aa 14.72±7.71Aa of P. tuberculatum, and found that extracts obtained using
12.5 32.05±3.71Aa 27.89±11.40Aa 29.49±6.03Aa solvents of lower polarity were more effective. Unlike Lima
%OR et al. (2014), we found that the strongest acaricidal activity
6.25 27.53±9.36 Aa
20.56±8.09 Aa
23.87±2.79Aa
of P. tuberculatum was produced by the leaf extracts, which
3.12 14.36±3.35Ab 26.27±8.62Aa 20.92±12.44Aa
can be due to the extraction method or solvent polarity (Silva
1.56 13.27±9.30 Ab
11.38±6.33 Aa
20.50±12.25Aa
et al. 2009).
50 55.63±12.88Aa 20.82±5.15Ba 55.63±4.26Aa
The extract from the fruits of P. tuberculatum was the
25 44.37±6.17 Ab
14.68±4.26 Ba
38.57±7.38Ab
most effective in reducing the egg hatching rate in this study,
12.5 1.02±0.59Cd 15.02±4.46Ba 55.97±4.69Aa yet Chagas et al. (2012) reported a 91.66% egg reduction
%HR
6.25 1.02±0.59 Cd
20.82±7.26 Ba
56.97±4.69Aa for a 10% (≈100 mg mL-1) concentration of P. tuberculatum
3.12 21.50±2.96Bc 21.16±2.71Ba 51.54±9.23Aa leaf extract, while at 5% (≈50 mg mL-1) the efficacy was only
1.56 18.77±5.25Bc 3.75±2.71Cb 54.61±8.59Aa 58.61%, close to that observed in our study. The efficacy
50 16.03±8.00 Bb
31.92±3.72 Ab
14.41±2.44Bb
of fruit extracts of P. tuberculatum on R. microplus females
was of 11.40% using ethanol as solvent, but was 100% and
25 20.29±2.93Bb 32.97±2.63Ab 24.09±4.14Ba
96.20% using hexane and ethyl ether, respectively, all at a
12.5 35.26±0.76 Aa
33.16±3.46 Ab
14.45±2.41Bb
ER concentration of 75 mg mL-1 in the adult immersion test
6.25 37.85±4.80Aa 31.61±3.93Bb 15.22±1.65Cb (Lima et al. 2014). The latter authors measured an LC50 of
3.12 33.85±3.61 Aa
32.20±3.12 Ab
17.55±5.6Bb 2.73 mg mL-1 for the ethanol extract of P. tuberculatum fruits
1.56 34.62±3.69Ba 45.08±2.21Aa 16.40±0.64Cb on R. microplus larvae, close to that observed in this study.
50 68.38±15.78 Aa
37.03±7.34 Ba
71.57±4.80Aa Santos et al. (2015) assessed the in vitro effect of the
25 59.98±5.78 Aa
34.96±5.20 Ba
52.48±8.16 Ab
aqueous extract, hydroalcoholic extract, concentrated
12.5 30.45±1.51Bb 34.60±6.82Ba 71.49±4.75Aa hydroalcoholic extract and essential oil of lemongrass
E (Cymbopogon wynterianus) on R. microplus larvae and engorged
6.25 25.33±9.48Cb 37.65±7.75Ba 69.97±3.25Aa
females. They found that the samples had acaricidal action,
3.12 33.24±7.12 Bb
36.48±6.14 Ba
65.38±10.96Aa
mainly the essential oil, because starting at a concentration of
1.56 31.71±7.28Bb 11.07±4.36Bb 67.64±1.26Aa
12.50% it caused 100% larval mortality, and at 25% it had

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an efficacy index of 100% in the test with engorged females, 1) for financial support and research scholarships, the
a better result than observed in our study. Fundação de Amparo ao Desenvolvimento das Ações Científicas e
Other Piper species have been evaluated for control of R Tecnológicas e à Pesquisa do Estado de Rondônia/ Coordenação
microplus larvae and adults. In vitro tests showed the toxic de Aperfeiçoamento de Pessoal de Nível Superior (FAPERO/
potential of the hexane extract and essential oil of the leaves CAPES) for one research scholarship, Fundação de Amparo à
of P. aduncum against R. microplus larvae and adults (Silva et Pesquisa do Estado do Amazonas (FAPEAM) for one doctoral
al. 2009). In an evaluation of essential oils of P. amalago, P. grant, and Empresa Brasileira de Pesquisa Agropecuária
mikanianum and P. xylosteoides, the highest mortality of R. (Embrapa - SEG 02.14.00.004.00.00) for financial support.
microplus larvae was observed for P. mikanianum (LC50=2.33
µL mL-1), followed by P. xylosteoides, while P. amalago showed REFERENCES
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larvae and engorged females of Rhipicephalus microplus. The hispidum H. B. K. Acta Amazonica, 38: 733–742.
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followed by the extracts of stems and leaves. Against engorged Artemisia annua ethanolic extract: prevention, simultaneous
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