Potential role of ATP-binding cassette transporters against acaricides in the brown dog tick Rhipicephalus sanguineus sensu lato

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1 Medical and Veterinary Entomology (2014), doi: /mve Potential role of ATP-binding cassette transporters against acaricides in the brown dog tick Rhipicephalus sanguineus sensu lato C. CAFARCHIA 1, D. PORRETTA 2, V. MASTRANTONIO 2, S. EPIS 3, D. SASSERA 4, R. I AT TA 1, D. IMMEDIATO 1, R. A. N. RAMOS 1, R. P. L I A 1, F. DANTAS-TORRES 1,5, L. KRAMER 6, S. URBANELLI 2 and D. OTRANTO 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 LC 50 doses and 95% confidence intervals (CIs) estimated by mortality data using probit analysis were p.p.m. (95% CI ) for fipronil and 3741 p.p.m. (95% CI ) for ivermectin. The pre-exposure of larvae to a sub-lethal dose of cyclosporine A reduced the LC 50 dose of fipronil to p.p.m. (95% CI ) and that of ivermectin to 167 p.p.m. (95% CI ), 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, Rome, Italy. Tel.: ; Fax: ; sandra.urbanelli@uniroma1.it These authors contributed equally to this paper The Royal Entomological Society 1

2 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, 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/m 2 ; 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

3 ABC transporters in R. sanguineus 3 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 et al., 2003) to estimate LC 50 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 LC 50 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 (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 LC 50 doses and 95% CIs estimated by mortality data using probit analysis were p.p.m. (95% CI ) for FIP and 3741 p.p.m. (95% CI ) for IVM. The pre-exposure of larvae to a sub-lethal dose of the ABC transporter inhibitor CsA reduced the LC 50 dose of FIP to p.p.m. (95% CI ) and that of IVM to 167 p.p.m. (95% CI ), 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 ( 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 ( 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

4 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 LC 50, p.p.m. (95% CI) SR (95% CI) Goodness-of-fit Equality Parallelism Fipronil ± ( ) 9.75 (5) 516 (2) (1) Fipronil + CsA ± ( ) ( ) (5) Ivermectin ± ( ) (4) 383 (2) 180 (1) Ivermectin + CsA ± (15 449) ( ) 8.55 (4) Chi-squared probability, < LC 50, 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

5 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: /mve 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. References Bouyer, F., Hamadou, S., Adakal, H., Lancelot, R., Stachurski, F., Belem, A.M. & Bouyer, J. (2011) Restricted application of insecticides: a promising tsetse control technique, but what do the farmers think of it? PLoS Neglected Tropical Diseases, 5, e1276. Buss, D.S. & Callaghan, A. (2008) Interaction of pesticides with p-glycoprotein and other ABC proteins: a survey of the possible importance to insecticide, herbicide and fungicide resistance. Pesticide Biochemistry and Physiology, 90, Buss, D.S., McCaffery, A.R. & Callaghan, A. (2002) Evidence for p-glycoprotein modification of insecticide toxicity in mosquitoes of the Culex pipiens complex. Medical and Veterinary Entomology, 16, Calvitti, M., Moretti, R., Porretta, D., Bellini, R. & Urbanelli, S. (2009) Effects on male fitness of removing Wolbachia infections from the mosquito Aedes albopictus. Medical and Veterinary Entomology, 23, Dantas-Torres, F., Figueredo, L.A. & Otranto, D. (2011) Seasonal variation in the effect of climate on the biology of Rhipicephalus sanguineus in southern Europe. Parasitology, 138, Dantas-Torres, F., Chomel, B.B. & Otranto, D. (2012) Ticks and tick-borne diseases: a One Health perspective. Trends in Parasitology, 28, Dantas-Torres, F., Latrofa, M.S., Annoscia, G., Giannelli, A., Parisi, A. & Otranto, D. (2013) Morphological and genetic diversity of Rhipicephalus sanguineus sensu lato from the New and Old Worlds. Parasites & Vectors, 6, 213. Deeley, R.G., Westlake, C. & Cole, S.P.C. (2006) Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins. Physiological Reviews, 86, Dermauw, W. & Van Leeuwen, T. (2014) The ABC gene family in arthropods: comparative genomics and role in insecticide transport and resistance. Insect Biochemistry and Molecular Biology, 45, Epis, S., Porretta, D., Mastrantonio, V. et al. (2014) ABC transporters are involved in defence against permethrin insecticide in the malaria vector Anopheles stephensi. Parasites & Vectors, 7, 349. Food & Agriculture Organization (1984) Resistance Management and Integrated Parasite Control in Ruminants Guidelines, Module 1 Ticks: Acaricide Resistance: Diagnosis, Management and Prevention. FAO, Animal Production and Health Division, Rome. Fernandes, F.F., D Alessandro, W.B.D. & Freitas, E.P.S. (2008) Toxicity of extract of Magonia pubescens (Sapindales: Sapindaceae) St. Hil. to control the brown dog tick, Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae). Neotropical Entomology, 37, Jones, P.M. & George, A.M. (2005) Multidrug resistance in parasites: ABC transporters, P-glycoproteins and molecular modelling. International Journal for Parasitology, 35,

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