Medical and Veterinary Entomology (2014), doi: 10.1111/mve.12093
Potential role of ATP-binding cassette transporters
against acaricides in the brown dog tick Rhipicephalus
sanguineus sensu lato
C.
S.
R.
S.
C A F A R C H I A 1† , D. P O R R E T T A 2† , V. M A S T R A N T O N I O 2 ,
E P I S 3 , D. S A S S E R A 4 , R. I A T T A 1 , D. I M M E D I A T O 1 ,
A. N. R A M O S 1 , R. P. L I A 1 , F. D A N T A S- T O R R E S 1,5 , L. K R A M E R 6 ,
U R B A N E L L I 2 and D. O T R A N T O 1
1
Department of Veterinary Medicine, University ‘Aldo Moro’ of Bari, Bari, Italy, 2 Department of Environmental Biology, University
of Rome ‘La Sapienza’, Rome, Italy, 3 Department of Veterinary Science and Public Health, University of Milan, Milan, Italy,
4
Department of Biology and Biotechnology, University of Pavia, Pavia, Italy, 5 Department of Immunology, Centro de Pesquisas
Aggeu Magalhães (Fiocruz), Recife, PE, Brazil and 6 Department of Animal Production, University of Parma, Parma, Italy
Abstract. ATP-binding cassette (ABC) transporters have been shown to be involved
in pesticide detoxification in arthropod vectors and are thought to contribute to the
development of drug resistance. Little is currently known about the role they play in
ticks, which are among the more important vectors of human and animal pathogens.
Here, the role of ABC transporters in the transport of fipronil and ivermectin acaricides
in the tick Rhipicephalus sanguineus (Ixodida: Ixodidae) was investigated. Larvae were
treated with acaricide alone and acaricide in combination with a sub-lethal dose of the
ABC transporter inhibitor cyclosporine A. The LC50 doses and 95% confidence intervals
(CIs) estimated by mortality data using probit analysis were 67.930 p.p.m. (95% CI
53.780–90.861) for fipronil and 3741 p.p.m. (95% CI 2857–4647) for ivermectin. The
pre-exposure of larvae to a sub-lethal dose of cyclosporine A reduced the LC50 dose
of fipronil to 4.808 p.p.m. (95% CI 0.715–9.527) and that of ivermectin to 167 p.p.m.
(95% CI 15–449), which increased toxicity by about 14- and 22-fold, respectively.
The comparison of mortality data for each separate acaricide concentration showed the
synergic effect of cyclosporine A to be reduced at higher concentrations of acaricide.
These results show for the first time a strong association between ABC transporters and
acaricide detoxification in R.sanguineus s.l.
Key words. Rhipicephalus sanguineus sensu lato, ABC transporters, cyclosporine A,
fipronil, ivermectin, tick-borne diseases.
Introduction
Pesticides remain among the most important tools used to
control arthropod vectors of many human and animal pathogens.
Different chemicals have been developed for this purpose,
including new generations of insecticides and acaricides, as
well as new formulations of existing compounds (Yu, 2008).
Concern about the environmental problems associated with the
use of pesticides (e.g. the accumulation of toxic compounds in
the environment and in the food web, and the disappearance
of non-target fauna) and the development of drug resistance in
target populations have increased the need for new alternative
Correspondence: Sandra Urbanelli, Department of Environmental Biology, University of Rome ‘La Sapienza’, 00185 Rome, Italy.
Tel.: + 39 06 4991 7820; Fax: + 39 06 4991 7820; E-mail: [email protected]
† These authors contributed equally to this paper.
© 2014 The Royal Entomological Society
1
2 C. Cafarchia et al.
or complementary control strategies (Otranto & Wall, 2008;
Calvitti et al., 2009). Furthermore, it is necessary to develop
strategies to increase the effectiveness of existing chemicals
against target pest populations (Porretta et al., 2007; Bouyer
et al., 2011).
Recent studies have shown the involvement of ATP-binding
cassette (ABC) transporters in pesticide detoxification in arthropod vectors (Buss & Callaghan, 2008; Dermauw & Van
Leeuwen, 2014). These ATP-dependent proteins have been
found in both prokaryote and eukaryote organisms and may
transport a large number of compounds across cellular membranes, including toxic metabolites and drugs. In humans, they
have been well characterized and widely studied as an important
component in the mechanism of drug resistance to chemotherapy. Amplification of these transporters is correlated with the
resistance that tumour cells can acquire to a broad range of drugs
[multidrug resistance (MDR)] (Buss & Callaghan, 2008). They
have also been shown to play a role in defence or resistance
against drugs in protozoa and helminths (Jones & George, 2005;
Sauvage et al., 2009). The detoxification activity of ABC transporters involves their reduction of the concentration of toxic
compounds inside cells, which they effect by preventing the
entry of these substances into or by promoting their exit from the
cell (Deeley et al., 2006). Therefore, ABC transporters may represent the cell’s first line of defence against exogenous cellular
toxic products. As they act on a wide spectrum of chemical products, ABC multidrug transporters may negatively affect the efficiency of pest control, reducing the effectiveness of pesticides
by their detoxification action and/or leading to the appearance
of multiple resistance. The inhibition of ABC-multidrug transporters has been shown to improve the performance of chemical compounds (Buss et al., 2002; Porretta et al., 2008). Indeed,
the inhibition of this cellular defence mechanism could increase
the susceptibility of organisms to pesticides and consequently
reduce the dose and frequency of application required.
Despite the growing interest in ABC transporters, at present
this detoxification mechanism has not been investigated in ticks,
with the exception of the cattle tick Rhipicephalus (Boophilus)
microplus (Pohl et al., 2011, 2012). Ticks are among the most
important arthropod vectors of human and animal pathogens.
They can transmit a wide range of viruses, bacteria and protozoa
(Dantas-Torres et al., 2012). Several tick species may survive
under different climatic conditions and ecological contexts and
have been suggested to increase their diffusion as a consequence
of human activities and global climate change (Dantas-Torres
et al., 2012; Porretta et al., 2013). Among ixodid ticks, Rhipicephalus sanguineus sensu lato is of great interest, being one
of the most widespread ticks in the world (Dantas-Torres et al.,
2013). This ixodid tick is a recognized vector of pathogens causing tick-borne diseases (TBDs) in dogs (e.g. Ehrlichia canis,
Babesia vogeli, Cercopithifilaria spp. and Hepatozoon canis)
and humans (e.g. Rickettsia conorii and Rickettsia rickettsii)
(Dantas-Torres et al., 2012).
In this study we aimed to investigate if ABC transporters are
involved in defence against the acaricides fipronil (FIP) and ivermectin (IVM) in R. sanguineus s.l. These are among the most
effective molecules currently used for tick control. Fipronil acts
by interrupting nerve impulses, leading to paralysis and death
in ticks. Ivermectin is a macrocyclic lactone derived from the
bacterium Streptomyces avermitilis, which interferes with nervous system and muscle function by enhancing inhibitory neurotransmission (Yu, 2008). To date, no evidence of resistance
to these acaricides has been shown in R. sanguineus s.l. populations (Miller et al., 2001). However, if ABC transporters are
involved in the efflux of acaricide from the cell, their inhibition should lead to a higher intracellular concentration of acaricide and to higher mortality (Pohl et al., 2011, 2012; Epis et al.,
2014). Therefore, in order to assess the possible association
between ABC transporters and the transport of FIP and IVM,
we performed bioassays with acaricide alone and in combination with a sub-lethal dose of cyclosporine A (CsA), an ABC
transporter inhibitor.
Materials and methods
Tick strains
Rhipicephalus sanguineus s.l. specimens were collected from
dogs in Putignano (40∘ 50′ N, 17∘ 07′ E; 375 m a.s.l.) in the
province of Bari, Italy, and identified based on morphological
and molecular features as Rhipicephalus sp. 1 (Dantas-Torres
et al., 2013). Female ticks were collected from clinically healthy
dogs which had never been exposed to acaricides, and maintained at the Parasitology Unit of the Department of Veterinary Medicine, University of Bari under controlled conditions
of temperature (27 ± 1 ∘ C), relative humidity (RH 80 ± 5%)
and photoperiod (LD 12 : 12 h) to lay eggs for 16 days. After
28 days, newly emerged larvae were obtained and were allowed
to feed on rabbits until detachment as previously described
(Dantas-Torres et al., 2011). Then, they were maintained under
controlled conditions (as above) to moult into nymphs. Newly
moulted nymphs were allowed to feed on rabbits until detachment and then maintained under controlled conditions (as above)
to moult into adults, which were placed (20 males and 20
females) on rabbits for feeding and mating. Then, engorged
females were transferred to controlled conditions (as above) to
lay eggs. In this way ticks were maintained for several generations. All procedures were carried out according to the guidelines for animal experimentation and were approved by the University of Bari (protocol no. 9/12). Bioassays were performed
using 14-day-old larvae (from the third to sixth generations) and
according to procedures standardized by the UN Food and Agriculture Organization (FAO, 1984).
Laboratory bioassays
Susceptibility of larvae to FIP and IVM. The larval packet
test (LPT) was used to investigate IVM and FIP lethal concentrations according to the FAO (1984), with some modifications
to improve practicality without compromising efficiency
(Fernandes et al., 2008; Senra et al., 2013). Specifically, each
packet contained 20 larvae and was composed of filter paper
measuring 2 × 4 cm (Labor 67 g/m2 ; Tecnochimica Moderna
Srl, Rome, Italy). Ivermectin (Sigma-Aldrich Srl, Milan, Italy)
was diluted at 1% in a 2 : 1 (v/v) trichloroethylene (TCE)
and olive oil vehicle (IVM stock solution). The IVM stock
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12093
ABC transporters in R. sanguineus
solution was diluted to six concentrations ranging from
100 p.p.m. to 8000 p.p.m. (i.e. 100, 1000, 2000, 4000, 6000
and 8000 p.p.m.) in the same vehicle. A 1% stock solution of
technical grade FIP (97%; Sigma-Aldrich Srl) was prepared
in acetone (100%) and seven dilutions ranging from 10 p.p.m.
to 100 p.p.m. (10, 12.5, 25, 50, 60, 80 and 100 p.p.m.) were
then prepared using the same vehicle as for IVM. A volume
of 100 𝜇L of each IVM or FIP dilution was applied over the
surface of the filter paper. The control group was exposed
to filter paper treated only with the organic solvents without
acaricide. The papers were maintained at room temperature for
6 h to allow TCE and acetone evaporation and were then formed
into packets (FAO, 1984). The bioassay for each acaricide was
composed of seven (IVM) or eight (FIP) groups of larvae (i.e.
one for each dilution and one as a control). Each group was
composed of three subgroups of 20 larvae, for a total of 60
larvae. Each bioassay was repeated three times; thus a total of
180 larvae for each dilution or control were tested. The larval
packets were sealed and incubated at 27 ± 1 ∘ C and RH 80 ± 5%
for 24 h, after which the mortality rate was determined. Only
larvae capable of moving spontaneously or when mechanically
stimulated with a needle were considered to be alive.
Susceptibility of larvae to CsA and detection of sub-lethal
dose. Bioassays were performed using the larval immersion test
(LIT) (Pohl et al., 2011). Briefly, CsA (Sigma-Aldrich Srl) was
diluted to 500 μm in 1% ethanol containing 0.02% Triton-X 100
(CsA stock solution). Then the CsA stock solution was diluted
to five concentrations ranging from 5 μm to 120 μm (i.e. 5, 15,
30, 60 and 120 μm) in the same vehicle. Volumes of 0.5 mL of
each CsA dilution or vehicle alone (control) were put into glass
tubes and approximately 60 larvae were added to each tube.
Immediately after the addition of larvae, the tube was closed
and shaken vigorously for 1 min and then gently for 10 min. The
tube was then opened and the larvae were placed on filter papers
(i.e. 20 larvae per single packet) impregnated with 50 𝜇L sterile
water. The larval packets were sealed and incubated at 27 ± 1 ∘ C
and RH 80 ± 5% for 24 h, after which the mortality rate was
determined as reported above. Each experiment was repeated
three times.
Susceptibility to IVM and FIP of larvae pre-exposed to a
sub-lethal dose of CsA. Before they were submitted to LPTs
with acaricide, 60 larvae for each acaricide dilution were
exposed to predetermined sub-lethal doses of CsA by LIT as
described above. Specifically, larvae were immersed in 0.5 mL
CsA and then placed on filter papers impregnated with each of
the IVM and FIP dilutions. The control group of 60 larvae was
immersed in 0.5 mL of CsA solvent and then exposed to the
filter paper treated with 100 𝜇L of the IVM or FIP solvent. The
LPT was performed as reported above and each experiment was
repeated three times.
Data analysis
Larval mortality data were subjected to probit regression
analysis as implemented in Polo Plus software (Robertson
3
et al., 2003) to estimate LC50 values with 95% confidence intervals (CIs). To estimate the effect on larval mortality of the ABC
transporter inhibitor at a sub-lethal dose, the synergism ratio
was calculated by the LC50 estimates of bioassays with acaricide
alone and in combination with CsA. The same software was used
to test the hypotheses of equality (equal slopes and intercepts)
and parallelism (equal slopes) of the regression lines from the
treatments with, respectively, acaricide alone and acaricide plus
CsA. Finally, we compared mortality data for larvae treated with
acaricide alone and larvae treated with acaricide plus CsA at
each concentration used and tested the significance of any differences using Student’s t-test in R Version 2.6.2 (R Development
Core Team, 2008).
Results
The sub-lethal dose of CsA detected by LIT was 30 μm. At this
concentration, we observed a mortality rate of approximately
2% in the control groups (Figure S1, online). Plots of larval
mortality × log (dose) and regression lines for FIP and IVM
alone and in combination with CsA are reported in Fig. 1. The
mortality data observed in bioassays with acaricides alone and
acaricides plus CsA fitted the probit dose–response model well
(chi-squared goodness-of-fit test, P > 0.05), although the 95%
CI for bioassays with acaricides plus CsA were higher than
those estimated for bioassays with acaricides alone as a result
of the high mortality (mean mortality: ∼ 50%) observed at the
lower acaricide concentrations (Fig. 1). The LC50 doses and
95% CIs estimated by mortality data using probit analysis were
67.930 p.p.m. (95% CI 53.780–90.861) for FIP and 3741 p.p.m.
(95% CI 2857–4647) for IVM. The pre-exposure of larvae to a
sub-lethal dose of the ABC transporter inhibitor CsA reduced
the LC50 dose of FIP to 4.808 p.p.m. (95% CI 0.715–9.527) and
that of IVM to 167 p.p.m. (95% CI 15–449), which increased
toxicity by about 14- and 22-fold, respectively.
The hypotheses of equality (equal slopes and intercepts) and
parallelism (equal slopes) of the regression lines from the
treatment with acaricide alone and acaricide plus CsA were
rejected for both FIP and IVM acaricides (all chi-squared tests,
P < 0.05) (Table 1). As shown in Fig. 2, CsA increased the
mortality of larvae treated with FIP for each concentration
used, although to a lesser degree at higher concentrations
(80–100 p.p.m.) (Fig. 2). By contrast, the addition of CsA to
IVM led to a significant increase in larval mortality at lower
concentrations of IVM (100–4000 p.p.m.; P < 0.05), but not at
higher concentrations (6000 p.p.m. and 8000 p.p.m.; P > 0.05).
Discussion
The use of compounds known to directly or indirectly disrupt
ABC transporter-mediated transport of drugs (chemosensitizers) represents a viable approach to investigating the association between ABC transporter activity and transport and/or
resistance to pesticides in several arthropod species. If ABC
transporters are involved in defence against pesticides, a higher
mortality rate can be expected in larvae treated with pesticide
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12093
4 C. Cafarchia et al.
Fig. 1. Larval mortality × log (dose) plots and regression lines for: (A) fipronil alone (black circles) and fipronil + cyclosporine A (CsA) (grey circles),
and (B) ivermectin alone (black circles) and ivermectin + CsA (grey circles). Cyclosporine A was used at 30 μm.
Table 1. Toxicity of acaricides and acaricides in conjunction with an ATP-binding cassette (ABC) transporter inhibitor.
𝜒 2 (d.f.)
Acaricide
Slope, mean± SE
LC50 , p.p.m. (95% CI)
SR (95% CI)
Goodness-of-fit
Equality
Parallelism
Fipronil
Fipronil + CsA
Ivermectin
Ivermectin + CsA
1.928 ± 0.177
1.220 ± 0.149
2.668 ± 0.165
0.532 ± 0.081
67.930 (53.780–90.861)
4.808 (0.715–9.527)
3741 (2857–4647)
167 (15–449)
—
14.127 (8.688–22.972)
—
22.350 (9.414–53.062)
9.75 (5)
13.37 (5)
17.90 (4)
8.55 (4)
516 (2)∗
—
383 (2)∗
—
10–63 (1)∗
—
180 (1)∗
—
∗Chi-squared probability, < 0.05.
LC50 , 95% confidence intervals (95% CIs) and slopes are estimated from mortality data by probit analysis.
95% CI, 95% confidence interval; CsA, cyclosporine A; SE, standard error; SR, synergism ratio.
plus an ABC transporter inhibitor than in those treated with
pesticide alone (Buss & Callaghan, 2008; Dermauw & Van
Leeuwen, 2014; Epis et al., 2014). In this study, the ABC transporter inhibitor CsA and the acaricides FIP and IVM were used
to treat 14-day-old larvae of R. sanguineus s.l. The mortality data
observed confirm the above hypothesis, thereby supporting the
suggestion that ABC transporters play a role in defence against
FIP and IVM acaricides. Indeed, we observed increased mortality rates of about 14- and 22-fold for FIP and IVM, respectively,
in larvae pre-exposed to the sub-lethal dose of CsA (Table 1,
Fig. 1).
Our results in R. sanguineus s.l. add new data on the involvement of ABC transporters in arthropod defence against FIP and
IVM, specifically for ABC transporters in ticks (Dermauw &
Van Leeuwen, 2014). An association between ABC transporters
and FIP has been shown only in a resistant strain of the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae)
(Dermauw & Van Leeuwen, 2014). Similarly, the association
between ABC transporters and macrocyclic lactones has been
investigated in arthropod species of medical or veterinary importance, such as Lepeophtheirus salmonis (Siphonostomatoida:
Caligidae), Pediculus humanus humanus (Phthiraptera: Pediculidae), Culex pipiens (Diptera: Culicidae), and Sarcoptes scabiei
(Sarcoptiformes: Sarcoptidae) (reviewed in Dermauw & Van
Leeuwen, 2014).
Among ticks, ABC transporters have been investigated only
in the cattle tick R. (B.) microplus. In this species, synergism
between CsA and IVM has been investigated and a synergism
ratio of 1.2 : 1.5 was recorded in resistant larvae, with no effect
in susceptible strains (Pohl et al., 2011, 2012, 2014). Although
any comparison of results obtained in different species should
be made with caution, it is worth noting that we found a
synergism ratio of 22 between CsA and IVM in a susceptible
strain. The increased mortality observed, therefore, may indicate
a strong association between ABC transporters and FIP and IVM
detoxification.
Interestingly, by comparing mortality data for each individual concentration, we observed that the addition of CsA
to acaricides significantly increased larval mortality at lower
concentrations of acaricide, whereas at the highest concentrations a reduced effect of CsA was observed. Cell defence
against harmful xenobiotics is known to be based on the
activity of several detoxifying enzymes other than ABC
transporters, such as cytochrome P450, carboxylesterases
and glutathione s-transferases (GSTs) or uridine diphosphate
(UDP)-glycosyltransferases, which chemically modify toxic
compounds (Xu et al., 2005). Their activity may therefore
account for the minimal effect of CsA when IVM and FIP are
used in high doses. Future studies that aim to identify the genes
of the different detoxifying enzymes involved and their expression profiles after acaricide treatment may help to elucidate the
factors underlying the dose-dependent effect observed.
In conclusion, this study provides evidence that R. sanguineus
s.l. larvae have ABC transporters, which are capable of
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12093
ABC transporters in R. sanguineus
5
Fig. 2. Mortality rates in larvae treated with: (A) fipronil alone and fipronil + cyclosporine A (CsA), and (B) ivermectin alone and ivermectin + CsA.
Data are shown as mean ± standard deviation. Cyclosporine A was used at 30 μm. *, significant differences between treatment with acaricide alone and
treatment with acaricide + CsA (Student’s t-test, P < 0.05).
protecting them to a certain extent from FIP and IVM activity.
The inhibition of pest-detoxifying mechanisms such as ABC
transporters may represent an effective strategy for improving
the efficacy of chemical control and reducing the environmental
impact related to the widespread use of chemicals. Certainly, the
pioneer study by Pohl et al. (2011) in R. (B.) microplus showed
the feasibility of this strategy in ticks. Further studies are
required to better elucidate the roles of these ABC transporters
and their inhibitors in the chemical control of tick infestations
in animals and humans.
Supporting Information
Additional Supporting Information may be found in the
online version of this article under the DOI reference: DOI:
10.1111/mve.12093
Figure S1. Mortality rate of larvae treated with cyclosporine A.
Acknowledgements
Funding for this work was provided by the Ministero
dell’Istruzione, dell’Università e della Ricerca (PRIN 2010).
The authors are grateful to Professor Claudio Genchi, Department of Veterinary Science and Public Health, University of
Milan, Milan, Italy, for his suggestions and comments on the
manuscript.
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Accepted 7 November 2014
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12093