Parasitol Res
DOI 10.1007/s00436-013-3527-8
ORIGINAL PAPER
Investigation of activity of monoterpenes
and phenylpropanoids against immature stages
of Amblyomma cajennense and Rhipicephalus sanguineus
(Acari: Ixodidae)
Tatiane Oliveira Souza Senra & Fernanda Calmon & Viviane Zeringóta &
Caio Márcio Oliveira Monteiro & Ralph Maturano & Renata da Silva Matos & Diego Melo &
Geovany Amorim Gomes & Mario Geraldo de Carvalho & Erik Daemon
Received: 22 June 2013 / Accepted: 26 June 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract The objective of this study was to assess the
acaricidal activity of carvacrol, thymol, eugenol, and (E)-
cinnamaldehyde on unengorged larvae and nymphs of
Amblyomma cajennense and Rhipicephalus sanguineus,
using the modified larval packet test. Carvacrol, eugenol,
and (E)-cinnamaldehyde were tested at concentrations of
2.5, 5.0, 10.0, 15.0, and 20.0 μl/ml, while thymol was tested
at concentrations of 2.5, 5.0, 10.0, 15.0, and 20.0 mg/ml, in
all cases with 10 repetitions per treatment. For the A.
cajennense larvae, mortality rates caused by carvacrol, thy-
mol, eugenol, and (E)-cinnamaldehyde at the lowest concen-
tration were 45.0, 62.7, 10.2, and 81.6 %, respectively,
reached 100 % at the concentration of 5.0 μl/ml for carvacrol
and (E)-cinnamaldehyde and 5.0 mg/ml for thymol, while
this mortality was observed at 15.0 μl/ml for eugenol. For the
nymphs of this species, carvacrol and thymol caused 100 %
mortality starting at a concentration of 5.0 μl/ml and 10.0
mg/ml, respectively, while eugenol caused 100 % mortality
at 20.0 μl/ml and the mortality caused by (E)-cinnamaldehyde
T. O. S. Senra (*) : F. Calmon : V. Zeringóta : R. da Silva Matos :
D. Melo : E. Daemon
Pós-Graduação em Ciências Biológicas - Comportamento e
Biologia Animal, Universidade Federal de Juiz de Fora,
Juiz de Fora, Brasil
e-mail: tatiosouza@ig.com.br
C. M. O. Monteiro : R. Maturano
Pós-Graduação em Ciências Veterinárias, Universidade Federal
Rural do Rio de Janeiro, Seropédica, Brasil
G. A. Gomes : M. G. de Carvalho
Departamento de Química,
Universidade Federal Rural do Rio de Janeiro,
Seropédica, Brasil
did not exceed 64 %. In the tests with R. sanguineus larvae, the
lowest concentration of carvacrol and (E)-cinnamaldehyde
resulted in 100 % mortality, while this percentage was ob-
served starting at 10.0 μl/ml for eugenol. For nymphs, carvacrol
and thymol at the smallest concentration caused 100 % lethality,
unlike the results for eugenol and (E)-cinnamaldehyde, where
100 % mortality was only observed starting at the concentration
of 10.0 μl/ml. The results obtained indicate that the tested
substances have acaricidal activity on unengorged larvae and
nymphs of A. cajennense and R. sanguineus.
Introduction
The preferred hosts of the Cayenne tick, Amblyomma
cajennense (Fabricius, 1787), are horses and capybaras, but
due to its low specificity, mainly in the immature stages, this
parasite also afflicts other animals such as deer, cattle, small
rodents, canids, birds, and even humans (Guimarães et al.
2001; Souza et al. 2004; Martins et al. 2004). This ectopar-
asite causes important economic damages to stock breeders
(Prata et al. 1996) and is also of great importance from the
standpoint of public health because it is the main tick that
afflicts humans in South America. In Brazil, Mexico,
Panama, and Colombia, it has been incriminated as a vector
of the bacterium Rickettsia rickettsii, the etiological agent of
spotted fever (Guglielmone et al. 2006; Labruna 2009).
Rhipicephalus sanguineus (Latreille, 1806), commonly
called the brown dog tick, has wide geographic distribution
and causes direct harm (blood spoliation) and indirect prob-
lems (transmission of pathogens) to dogs (Labruna 2004;
Dantas-Torres 2008; Labruna et al. 2009). This tick can also
transmit diseases to humans. In the Mediterranean region, it

has been indicated as a vector of the bacterium Rickettsia
conorii, the etiological agent of boutonneuse fever (Sousa and
Bacellar 2004; Matsumoto et al. 2005), while it is considered a
vector of R. rickettsii in the USA and Mexico (Bustamante and
Varela 1947; Demma et al. 2005). In Brazil, R. sanguineus is
also considered a potential vector of R. rickettsii to humans
(Pacheco et al. 2011).
Control of these two ectoparasites is mainly through
synthetic acaricides, but the indiscriminate and techni-
cally improper use of these substances causes problems,
such as human and animal intoxication and the selection
of resistant ticks (Kaaya et al. 1996; Borges et al.
2011). The employment of substances obtained from
plants is a promising alternative because it can be a
safer way to control these parasites, by reducing the
risks of environmental contamination and human and
animal intoxication (Koul et al. 2008; Borges et al.
2011; Kiss et al. 2012).
There has been a good deal of research into the activity of
plant essential oils as well as their main components. Among
the various substances investigated, the monoterpenes thymol
and carvacrol and the phenylpropanoids (E)-cinnamaldehyde
and eugenol have been indicated as particularly promising due
to their activity against bacteria, insects, and ticks/mites (Lee
et al. 2008; Ali et al. 2005; Pozzo et al. 2011; Shen et al. 2012).
The acaricidal activity of carvacrol, eugenol, and (E)-
cinnamaldehyde against unengorged larvae of Rhipicephalus
microplus (Canestrini, 1888) and Dermacentor nitens
(Neumann, 1897) has been demonstrated (Monteiro et al.
2012; Senra et al. 2013). In turn, the activity of thymol has
been shown against R. microplus, R. sanguineus, A.
cajennense and D. nitens (Monteiro et al. 2009, 2010;
Mendes et al. 2011; Daemon et al. 2012). However, this
monoterpene has not yet been tested in an ethanol formulation
on unengorged larvae and nymphs of A. cajennense and
unengorged nymphs of R. sanguineus. Therefore, the aim of
this study was to add to these earlier findings by assessing the
acaricidal activity of these substances on unengorged larvae
and nymphs of A. cajennense and R. sanguineus.
Material and methods
The experiments were performed at the Laboratório de
Artrópodes Parasitos (Arthropod Parasite Laboratory) of
Universidade Federal de Juiz de Fora, located in Juiz de Fora,
Minas Gerais, Brazil. The unengorged A. cajennense and R.
sanguineus larvae and nymphs came from colonies maintained
through artificial infestations of rabbits Oryctolagus cuniculus
(Linnaeus, 1758) (crossbreeds—California × New Zealand)
according to Neitz et al. (1971). The larvae tested were between
15 and 21 days old, while the nymphs were tested 15 days after
ecdysis.
Parasitol Res
The carvacrol and (E)-cinnamaldehyde tested were ac-
quired from Sigma-Aldrich, with 99.9 % purity. The thymol
(crystal form, 99.9 % purity) and eugenol (99.9 % purity)
were purchased from Henrifarma Químicos e Farmacêuticos
Ltda and Biodinâmica, respectively. Ethanol at 50° GL was
used to dissolve the carvacrol, thymol, and eugenol, while
ethanol at 70° GL was used for the (E)-cinnamaldehyde, due
to the inherent characteristics of each substance. Carvacrol,
eugenol, and (E)-cinnamaldehyde in all the experiments
were tested at concentrations of 2.5, 5.0, 10.0, 15.0, and
20.0 μl/ml, while the thymol was tested at concentrations
of 2.5, 5.0, 10.0, 15.0, and 20.0 mg/ml.
For the larvae, the larval packet test was used (Stone and
Haydock 1962), as modified by Monteiro et al. (2012), in which
approximately 50 larvae were placed on filter paper sheets
measuring 6×6 cm; after which, the sheets were folded in the
middle, and the edges were sealed with clips. Then, each side of
the resulting packets was moistened evenly with 90 μl of the test
solutions. In the control groups, the larvae were treated only
with the solvents that were used for each tested substance. There
were 10 repetitions for all groups. Thymol was not tested against
the R. sanguineus larvae since the activity of this monoterpene
in hydroethanolic formulation on unengorged larvae of this tick
has already been demonstrated by Daemon et al. (2012).
The same method was used with the unengorged nymphs,
except only five nymphs were placed in each packet. The
experimental groups were kept in a climate-controlled chamber
(27±1 °C and relative humidity (RH)>80 %), with a separate
chamber used for each substance, including the control groups.
The mortality was evaluated after 24 h by counting the
living and dead larvae and nymphs. The mortality was
expressed in percent: Mortality(%)=(total dead individuals/
total individuals)×100.
The statistical analysis was carried out with the Biostat
version 5.0 software (Ayres et al. 2007). The percent values
were transformed to √arcsine x and analyzed by analysis of
variance and the Tukey test (p<0.05). In the case of nonpara-
metric distributions, the values were compared by the Kruskal–
Wallis and Student–Newman–Keuls tests (p<0.05).
Results
In the tests with the A. cajennense larvae, the lowest concen-
tration of carvacrol, thymol, eugenol, and (E)-cinnamaldehyde
resulted in mortality rates of 45.0, 62.7, 10.2, and 81.6 %,
respectively. Carvacrol and (E)-cinnamaldehyde produced
100 % mortality starting at the concentration of 5.0 μl/ml,
while for thymol and eugenol, the lowest concentration that
caused 100 % mortality were 5.0 mg/ml and 15.0 μl/ml,
respectively (Table 1). The mortality rates obtained from the
lowest concentration of all the substances differed signifi-
cantly (p<0.05) from the control (Table 1).
Parasitol Res

Table 1 Mortality of A. cajennense larvae treated with different concentrations of carvacrol, thymol, eugenol, and (E)-cinnamaldehyde in
hydroethanolic formulations under laboratory conditions (27±1 °C and RH>80±10 %)

Carvacrol (μl/ml) Thymol (mg/ml) Eugenol (μl/ml) (E)-cinnamaldehyde (μl/ml)

Control 0.0 a±0.0a 0.0 a±0.0a 0.0 a±0.0a 0.0 a±0.0b

2.5 45.0 b±16.1 62.7 b±16.5 10.2 b±8.2 81.6 b±11.7
5.0 100.0 b±0.0 100.0 c±0.0 41.6 bc±23.4 100.0 b±0.0
10.0 100.0 b±0.0 100.0 c±0.0 87.2 c±9.3 100.0 b±0.0
15.0 100.0 b±0.0 100.0 c±0.0 100.0 c±0.0 100.0 b±0.0
20.0 100.0 b±0.0 100.0 c±0.0 100.0 c±0.0 100.0 b±0.0

Means followed by different letters in the same column differ significantly (p<0.05)
a
Control group: ethanol 50° GL
b
Control group: ethanol 70° GL

The results obtained from the tests with A. cajennense
nymphs (Table 2) demonstrated that carvacrol and thymol
caused 100 % mortality starting at concentrations of
5.0 μl/ml and 10.0 mg/ml, respectively. Eugenol only
attained this efficiency at the highest concentration tested
(20.0 μl/ml). In the groups treated with (E)-cinnamaldehyde,
the mortality did not exceed 64 %. In relation to the control
group, the mortality of the groups treated with carvacrol was
significantly different (p<0.05) at all concentrations, while
the same occurred with thymol starting at 5.0 mg/ml. For the
phenylpropanoids (E)-cinnamaldehyde and eugenol, signif-
icant differences were observed with the concentration of
10.0 μl/ml.
For the R. sanguineus larvae, carvacrol and (E)-
cinnamaldehyde both caused 100 % mortality at the lowest
concentration (Table 3). Due to the absence of variance in
these results, it was not possible to perform statistical anal-
ysis. In the groups treated with eugenol, only the lowest
concentration tested (2.5 μl/ml) did not produce significantly
different mortality (19.2 %) from the control group; at
5.0 μl/ml, the mortality was 97.8 % and reached 100 % at
the upper concentrations (Table 3).
In the tests with R. sanguineus nymphs, the monoter-
penes carvacrol and thymol caused 100 % lethality at all
the tested concentrations, but this same efficiency was
only observed starting at a concentration of 10.0 μl/ml
for the phenylpropanoids eugenol and (E)-cinnamaldehyde.
The groups treated with eugenol and (E)-cinnamaldehyde at
the lowest concentration (2.5 μl/ml) did not present a signif-
icant difference (p>0.05) in relation to the control group
(Table 4).
Discussion
Research has been conducted with substances of plant origin
into potential acaricidal activity due to the need to develop
new alternatives that can be used for integrated management
of ticks (Ferrarini et al. 2008; Monteiro et al. 2009; Mendes
et al. 2011; Chagas et al. 2012; Scoralik et al. 2012; Kiss
et al. 2012). The essential oils extracted from plants are
composed of various constituents that can be tested individ-
ually, which allows checking the activity of these isolated
substances on different organisms (Combrinck et al. 2011).

Table 2 Mortality of A. cajennense nymphs treated with different concentrations of carvacrol, thymol, eugenol, and (E)-cinnamaldehyde in
hydroethanolic formulations under laboratory conditions (27±1 °C and RH>80±10 %)

Carvacrol (μl/ml) Thymol (mg/ml) Eugenol (μl/ml) (E)-cinnamaldehyde (μl/ml)

Control 0.0 a±0.0a 0.0 a±0.0a 2.0 a±6.3a 0.0 a±0.0b

2.5 64.6 b±26.3 2.0 a±6.3 2.0 a±6.3 0.0 a±0.0
5.0 100.0 b±0.0 40.0 b±29.4 0.0 a±0.0 6.5 a±10.5
10.0 100.0 b±0.0 100.0 b±0.0 19.0 b±23.3 18.0 b±14.7
15.0 100.0 b±0.0 100.0 b±0.0 22.0 b±31.9 24.0 b±29.5
20.0 100.0 b±0.0 100.0 b±0.0 100.0 b±0.0 64.0 b±33.7

Means followed by different letters in the same column differ significantly (p<0.05)
a
Control group: ethanol 50° GL
b
Control group: ethanol 70° GL
 Parasitol Res

Table 3 Mortality of R. sanguineus larvae treated with different concen- that carvacrol was effective on larvae of R. microplus and D.
trations of carvacrol, eugenol, and (E)-cinnamaldehyde in hydroethanolic
nitens (Neumann, 1897), with 100 % mortality at the lowest
formulations under laboratory conditions (27±1 °C and RH>80±10 %)
concentration tested (2.5 μl/ml). Our results, showing high
Carvacrol (μl/ml) Eugenol (μl/ml) (E)-cinnamaldehyde sensitivity of unengorged A. cajennense and R. sanguineus
(μl/ml) larvae and nymphs, corroborate these earlier findings.
Control 0.0±0.0a 0.0 a±0.0a 0.0±0.0b
Thymol, a monoterpene isomer of carvacrol, also was
2.5 100.0±0.0 19.2 a±23.2 100.0±0.0
highly toxic from the lowest concentrations tested. This activ-
ity was expected, since this substance’s acaricidal activity has
5.0 100.0±0.0 97.8 b±4.6 100.0±0.0
already been demonstrated for others tick species at different
10.0 100.0±0.0 100.0 b±0.0 100.0±0.0
stages, including R. sanguineus and A. cajennense (Monteiro
15.0 100.0±0.0 100.0 b±0.0 100.0±0.0
et al. 2009; Mendes et al. 2011). However, for R. sanguineus,
20.0 100.0±0.0 100.0 b±0.0 100.0±0.0
thymol dissolved in ethanol has only been tested on
Means followed by different letters in the same column differ signifi- unengorged larvae (Daemon et al. 2012), so this article is the
cantly (p<0.05) first report for nymphs. With respect to A. cajennense, this
a
Control group: ethanol 50° GL monoterpene has already been tested in aqueous formulation,
b
Control group: ethanol 70° GL using dimethyl sulfoxide (1 %) as solubilization adjuvant
(Mendes et al. 2011), and the mortality of unengorged larvae
Among the substances tested here, carvacrol stands out as was greater than 90 % starting at the concentration of 10-
the most toxic since it resulted in high mortality rates among mg/ml. In the present study, the concentration of 5.0 mg/ml
larvae and nymphs of R. sanguineus and A. cajennense from resulted in 100 % death of the larvae, demonstrating that the
the lowest concentrations. This monoterpene’s acaricidal solubilization in ethanol enhanced the acaricidal activity of
activity has already been demonstrated for others tick species thymol on this stage. That finding has also been reported for
(Coskun et al. 2008; Dolan et al. 2009; Cetin et al. 2010; unengorged larvae of R. microplus (Scoralik et al. 2012) and
Senra et al. 2013). Cetin et al. (2009) evaluated the activity of R. sanguineus (Daemon et al. 2012). The latter authors attrib-
the essential oil of Origanum minutiflorum O. Schwarz & uted this enhancement effect to the better solubility of thymol
P.H. Davis on adults of Rhipicephalus turanicus Pomerantzev, in ethanol. For unengorged A. cajennense and R. sanguineus
1940 and observed 100 % mortality after 2 h of exposure. nymphs, this is the first report of the activity of thymol.
They attributed this to the presence of carvacrol in the oil’s As mentioned for carvacrol, essential oils of plants that
composition. The same observation was reported by Lage contain thymol in their composition also have been shown to
et al. (2013), who tested the oil of Lippia triplinervis have acaricidal activity. For example, Gomes et al. (2012) tested
Gardner on engorged R. microplus females. According to the essential oil of Lippia sidoides Cham., whose main constit-
Cetin et al. (2010), 3 h of exposure to this substance at a uent is thymol, and found high acaricidal activity on larvae of
concentration of 10.0 μl/L was sufficient to kill 93.3 % D. nitens and engorged females and larvae of R. microplus.
of adults of Hyalomma marginatum Kock, 1844. Dolan Eugenol is found in the oil of Syzygium aromaticum (L.)
et al. (2009), in tests involving spraying a 5 % solution of Merr. & L. M. Perry (clove) and plants of the Lauraceae
carvacrol on plants, found a significant reduction in nymphs and Lamiaceae families (Franz and Novak 2009). This
of Ixodes scapularis Say, 1821 and Amblyomma americanum phenylpropanoid repellent power has also been demonstrated
(Linnaeus, 1758) in the environment. Senra et al. (2013) found on nymphs of Ixodes ricinus (Linnaeus, 1758) (Del Fabbro

Table 4 Mortality of R. sanguineus nymphs treated with different concentrations of carvacrol, eugenol, and (E)-cinnamaldehyde in hydroethanolic
formulations under laboratory conditions (27±1 °C and RH>80±10 %)

Carvacrol (μl/ml) Thymol (mg/ml) Eugenol (μl/ml) (E)-cinnamaldehyde (μl/ml)

Control 2.0±6.3a 2.0±6.3a 2.0 a±6.3a 1.7a±5.3b

2.5 100.0±0.0 100.0±0.0 31.5 a±31.0 52.0 ab±24.9
5.0 100.0±0.0 100.0±0.0 82.3 b±31.9 88.3 bc±19.2
10.0 100.0±0.0 100.0±0.0 100.0 b±0.0 100.0 c±0.0
15.0 100.0±0.0 100.0±0.0 100.0 b±0.0 100.0 c±0.0
20.0 100.0±0.0 100.0±0.0 100.0 b±0.0 100.0 c±0.0

Means followed by different letters in the same column differ significantly (p<0.05)
a
Control group: ethanol 50° GL
b
Control group: ethanol 70° GL
Parasitol Res
and Nazzi 2008) and larvae of R. microplus and D. nitens
(Zeringóta et al. 2013). Martinez-Velazquez et al. (2011)
found acaricidal activity of the essential oils of Pimenta dioica
(Linnaeus) and Ocimum basilicum L. on R. microplus and
attributed this effect to the methyl eugenol and eugenol
contained in these oils. In turn, Monteiro et al. (2012) dem-
onstrated the deleterious action of eugenol, using the same
method and same concentrations as in the present study, on R.
microplus and D. nitens larvae and found mortality rates
above 99 % for R. microplus from the lowest concentration
(2.5 μl/ml). In the present study, similar mortality was only
observed starting at the concentration of 5.0 μl/ml for R.
sanguineus and 10.0 μl/ml for A. cajennense. Based on these
results, it can be inferred that R. sanguineus and A. cajennense
larvae are less sensitive to eugenol than are R. microplus
larvae, a fact also observed for thymol in previous studies
(Daemon et al. 2009; Mendes et al. 2011).
According to Senra et al. (2013), (E)-cinnamaldehyde has
acaricidal activity on R. microplus and D. nitens larvae.
These authors used the same method of present study and
observed mortality rates above 90 % at the concentration of
2.5 μl/ml. However, this phenylpropanoid was the only
substance that even at the highest concentration tested here
(20 μl/ml) did not cause 100 % mortality to A. cajennense
nymphs (maximum rate of 64 %). That fact demonstrates the
variation of sensitivity between different tick species and
different stages of the same species, thus indicating the need
for detailed studies of each substance.
The results obtained in this study indicate that the mono-
terpenes carvacrol and thymol and the phenylpropanoids
eugenol and (E)-cinnamaldehyde have deleterious activity
on unengorged larvae and nymphs of R. sanguineus and
A. cajennense. Hence, there is a need for new studies
involving other developmental stages of these ticks and
to investigate the synergism between substances, along
with tests under natural conditions to corroborate the
promising laboratory results, for future use of these sub-
stances as acaricides.
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