Accepted Manuscript Title: In vitro establishment of ivermectin-resistant Rhipicephalus microplus cell line and the contribution of ABC transporters on the resistance mechanism Author: Paula C. Pohl Danielle D. Carvalho Sirlei Daffre Itabajara da Silva Vaz Jr Aoi Masuda PII: DOI: Reference:
S0304-4017(14)00339-2 http://dx.doi.org/doi:10.1016/j.vetpar.2014.05.042 VETPAR 7280
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
21-2-2014 23-5-2014 31-5-2014
Please cite this article as: Pohl, P.C., Carvalho, D.D., Daffre, S., Jr, I.S.V., Masuda, A.,In vitro establishment of ivermectin-resistant Rhipicephalus microplus cell line and the contribution of ABC transporters on the resistance mechanism, Veterinary Parasitology (2014), http://dx.doi.org/10.1016/j.vetpar.2014.05.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title:
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In vitro establishment of ivermectin-resistant Rhipicephalus microplus cell line and the
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contribution of ABC transporters on the resistance mechanism
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Authors:
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Paula C. PohlA,C, Danielle D. CarvalhoB, Sirlei DaffreC, Itabajara da Silva Vaz JrA,D,*, Aoi
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MasudaA,E
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A
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Gonçalves, 9500, Prédio 43421, Porto Alegre, RS, 91501-970, Brazil.
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B
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Rua João de Lacerda Soares, 316, São Paulo, SP, 04707-010, Brazil.
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Paulo, Avenida Professor Lineu Prestes, 1374, São Paulo, SP, 05508-900, Brazil.
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Gonçalves, 9090, Porto Alegre, RS, 91540-000, Brazil.
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do Sul, Avenida Bento Gonçalves, 9500, Prédio 43421, Porto Alegre, RS, 91501-970, Brazil.
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E-mail address:
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Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento
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Departamento de Análises Especiais, SD&W Modelagem e Soluções Estratégicas Ltda.,
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Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São
Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Avenida Bento
Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande
Corresponding author: Tel.: + 55 (51) 33086078; Fax: + 55 (51) 33087309
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Abstract The cattle tick Rhipicephalus microplus is one of the most economically damaging
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livestock ectoparasites, and its widespread resistance to acaricides is a considerable challenge to
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its control. In this scenario, the establishment of resistant cell lines is a useful approach to
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understand the mechanisms involved in the development of acaricide resistance, to identify drug
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resistance markers, and to develop new acaricides. This study describes the establishment of an
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ivermectin (IVM)-resistant R. microplus embryonic cell line, BME26-IVM. The resistant cells
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were obtained after the exposure of IVM-sensitive BME26 cells to increasing doses of IVM in a
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step-wise manner, starting from an initial non-toxic concentration of 0.5 µg/mL IVM, and
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reaching 6 µg/mL IVM after a 46-week period. BME26-IVM cell line was 4.5 times more
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resistant to IVM than the parental BME26 cell line (lethal concentration 50 (LC50) 15.1 ± 1.6
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µg/mL and 3.35 ± 0.09 µg/mL, respectively). As an effort to determine the molecular
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mechanisms governing resistance, the contribution of ATP-binding cassette (ABC) transporter
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was investigated. Increased expression levels of ABC transporter genes were found in IVM-
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treated cells, and resistance to IVM was significantly reduced by co-incubation with 5 µM
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cyclosporine A (CsA), an ABC transporter inhibitor, suggesting the involvement of these
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proteins in IVM-resistance. These results are similar to those already described in IVM-resistant
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tick populations, and suggest that similar resistance mechanisms are involved in vitro and in
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vivo. They reinforce the hypothesis that ABC transporters are involved in IVM resistance and
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support the use of BME26-IVM as an in vitro approach to study acaricide resistance
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mechanisms.
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Keywords: ATP-binding cassette transporter, ivermectin resistance, Rhipicephalus microplus,
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tick cell line. 2 Page 2 of 26
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1. Introduction Rhipicephalus microplus is one of the most deleterious cattle ectoparasites worldwide. In
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addition to the direct effects of R. microplus infestation on milk, meat, and leather production,
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this tick species is the vector for the infectious agents that cause bovine babesiosis and
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anaplasmosis (Jonsson, 2006; Jonsson et al., 2008).
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The development of chemicals with acaricidal properties favored the adoption of these
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substances as the main method of cattle tick control (Guerrero et al., 2012). However, R.
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microplus has become resistant to most commercially available acaricides, thereby reducing the
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effectiveness of tick control (Klafke et al., 2006; Castro-Janer et al., 2011; Guerrero et al., 2012).
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Macrocyclic lactones, such as ivermectin (IVM), are increasingly used to control endo-
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and ectoparasites, including R. microplus (Fox, 2006; Guerrero et al., 2012). IVM activates
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glutamate-gated chloride ion channels in invertebrate nerve and muscle cells, causing the
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paralysis of peripheral motor function and death of the organism (Fox, 2006). However, recent
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reports concerning IVM-resistant cattle tick populations in Brazil (Martins and Furlong, 2001;
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Klafke et al., 2006), in Mexico (Perez-Cogollo et al., 2010), and in Uruguay (Castro-Janer et al.,
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2011) have emphasized the importance of understanding the mechanisms of acaricide resistance
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to improve tick control methods.
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Previous studies have associated ATP-binding cassette (ABC) transporters with drug
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resistance in nematodes (James and Davey, 2009), arthropods (Pohl et al., 2011, Atsumi et al.,
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2012), and cancer cells (Gillet et al., 2007). ABC transporters are integral membrane proteins
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expressed in all organisms, and are essential for several physiological processes (Holland and
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Blight, 1999). Certain ABC transporters are involved in the cellular detoxification of xenobiotics
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by pumping the drugs across membranes before they can reach their target site (Szakács et al.,
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2008). ABC transporters are able to transport drugs with diverse structures and functions,
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conferring a cellular phenotype termed “multidrug resistance” (Higgins, 2007). Recently, we demonstrated the association of ABC transporters with macrocyclic
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lactones (IVM, abamectin and moxidectin) and organophosphate resistance (Pohl et al., 2011;
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Pohl et al., 2012) in R. microplus. The results of these studies linked the participation of ABC
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transporters to drug resistance, via a multidrug detoxification mechanism.
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Currently, the routine detection of acaricide resistance is based on bioassay techniques,
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such as the larval packet test and the adult immersion test; however, bioassays are time-
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consuming and laborious (Drummond et al., 1973; Klafke et al., 2006). The development of
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more sensitive diagnostic methods based on molecular biology methodologies is a convenient
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alternative, such as the allele-specific PCR assay to detect a mutation in the para-type sodium
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channel gene, which identifies pyrethroid resistance in ticks (Guerrero et al., 2001). However,
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the development of more sensitive methods relies on understanding the mechanisms involved in
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resistance, which in turn depends on studying tick populations in which acaricide resistance has
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already been established in the field.
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In this sense, in vitro cell culture is an attractive alternative, because the experimental
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parameters can be more easily controlled, thereby reducing variability between experiments.
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Additionally, cell culture is a cost-effective approach to investigate drug resistance mechanisms,
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even before resistance has been established in field populations. Moreover, cell culture
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experiments allow the rapid screening of new drugs under development. For example, the
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selection of cancer cell lines for resistance to cytotoxic drugs has been a key element in the
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identification of mutations responsible for drug resistance (Edwards et al., 2008).
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Considering the importance of understanding the mechanisms of drug resistance to
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improve the detection and prevention of acaricide-resistant tick populations, we questioned if an
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acaracide resistant tick cell line selected in vitro has similar resistance mechanisms to those
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governing acaricide resistance in vivo, which would be a useful approach to study acaricide
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resistance. In this study, we report the in vitro selection of an IVM-resistant R. microplus cell
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line derived from the BME26 cell line (Esteves et al., 2008), and demonstrate the involvement of
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ABC transporters in the process of IVM resistance in these cells.
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2.1. Cell lines and maintenance
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An IVM-resistant cell line (BME26-IVM) was derived from drug-sensitive BME26 cells,
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which were originally isolated from R. microplus embryos (Esteves et al., 2008). The cell lines
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were grown as adherent monolayers in complete medium, as described by Esteves et al. (2008)
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(L-15B300 medium supplemented with 5% heat-inactivated Fetal Bovine Serum (FBS) (Gibco),
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10% Tryptose Phosphate Broth (TPB) (BD), penicillin (100 U/mL), streptomycin (100 mg/mL)
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(Gibco), and 0.1% bovine lipoprotein concentrate (ICN), pH 7.2). For BME26-IVM cells, the
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complete medium was supplemented with IVM (6 µg/mL) (Sigma) diluted from a stock solution
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of 2 mg/mL IVM in 50% methanol. The cells were grown at 34 °C in 25 cm2 plastic flasks
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(Falcon) in 5 mL of the medium, which was replaced every 7 days.
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2.2. Establishment of IVM-resistant cell line
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The BME26-IVM cells were derived from the parental BME26 cells (23rd passage) by
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continuous exposure to increasing concentrations of IVM in a step-wise manner. Every 4 weeks,
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the cells were harvested with a cell scraper and transferred into a new plastic flask containing the
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same concentration of IVM or into a flask containing a higher concentration of the drug. Starting
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from an initial non-toxic concentration of 0.5 µg/mL IVM, the cells were eventually able to grow
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in the presence of 6 µg/mL IVM after 46 weeks of selection.
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2.3. Growth curve and doubling time determination
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BME26 (40th passage) or BME26-IVM (38th passage) cells were seeded (5 x 105 cells) in
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25 cm2 plastic flasks in triplicate and cultured in 5 mL of complete medium. After a 4 h
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incubation period, the medium was replaced with fresh complete medium or complete medium
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supplemented with IVM. Every 7 days, viable cell density was determined by counting trypan
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blue (Sigma)-stained cells (0.04% trypan blue in phosphate buffered saline (145 mM NaCl, 80
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mM Na2HPO4, and 20 mM NaH2PO4)) using a Neubauer hemocytometer (Esteves et al., 2008)
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to generate a growth curve over a 28-day culture period.
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The cell doubling-times (dt) were calculated during exponential growth (7th to 28th days)
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using the equation dt = ln(2)/n, where n= [ln (final number of cells) – ln (initial number of
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cells)]/ [(final time) – (initial time)].
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2.4. Cytotoxicity assay
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The tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
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bromide) was used to evaluate the cytotoxicity of different IVM concentrations in the absence or
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presence of cyclosporine A (CsA), an ABC transporter inhibitor (Choi, 2005). BME26 (41st
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passage) or BME26-IVM (39th passage) cells were seeded (1x106 cells) in 25 cm2 plastic flasks
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in triplicate and incubated in complete medium. After a 4-h incubation period, the medium was
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replaced with fresh complete medium without IVM or with medium supplemented with IVM at a
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concentration of 1, 3, 6 or 9 µg/mL, with or without 5 µM CsA (predetermined non-toxic dose).
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After a 9-day incubation period, the cells were harvested with a cell scraper, and 1 mL of the cell
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suspension in each flask was transferred in triplicate to a 24-well plate (Nunc), and incubated for
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2 h to sediment the cells on the plate. The medium was replaced with 0.5 mL of MTT solution
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(0.5 mg/mL in cell medium), and the cells were incubated for an extra 90-min period at 34 °C.
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The MTT solution was removed and replaced with 0.5 mL of 0.002% HCl in isopropanol. The
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plate was further incubated for 5 min at room temperature to dissolve the dark blue formazan
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crystals, and 100 µL of solution was transferred to a 96-well plate. Optical density values were
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measured at a wavelength of 570 nm using a multiwell scanning spectrophotometer (Labsystems
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iEMS). The percentage of viable cells treated with IVM or IVM + CsA was normalized to the
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untreated cells, which were considered to be 100% viable. The lethal concentration 50 (LC50),
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defined as the concentration necessary to reduce cell survival by 50%, was calculated from dose-
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response curves in the presence or absence of CsA. The resistance ratio (RR) was estimated by
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dividing the LC50 of the BME26-IVM cells by that of the parental BME26 cells. The fold-
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reversal factor was estimated by dividing the LC50 of the cell line treated with IVM and without
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CsA by that of the cell line treated with both IVM and CsA.
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2.5. Light microscopy
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Cells were collected by scraping and were centrifuged onto microscope slides at 1,000 x
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g for 5 min using a cytocentrifuge (Fanem). The cells were air-dried, stained with Panoptico
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(Newprov) stain, and observed under oil immersion using a light microscope at ×1000
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magnification.
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2.6. Reverse transcription quantitative PCR (RT-qPCR)
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To evaluate the mRNA expression of ABC transporters, BME26 and BME26-IVM cells
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were incubated for 9 days in complete medium alone or complete medium with 6 or 9 µg/mL
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IVM. The cells were then collected by scraping and centrifuged at 5,000 x g for 5 min. Total
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RNA was extracted from the cells using TRIzol® reagent (Invitrogen) following the
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manufacturer’s recommendations. The RNA quantity was estimated spectrophotometrically at
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260 nm using a NanoDrop 1000 instrument (Thermo Fisher Scientific). One microgram of total
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RNA was treated with DNase I (Invitrogen) and reverse-transcribed using the High-Capacity
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cDNA Reverse Transcription Kit with random primers, according to the manufacturer’s
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recommendations (Applied Biosystems). RT-qPCR was performed to quantify the mRNA of
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ABC transporters using the Quantimix Easy SYBR Green Amplification Kit (Biotools) in a
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Mastercycler® ep realplex real-time PCR instrument (Eppendorf). The specific primers designed
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for gene amplification are listed in Table 1. Gene amplification of the 40-S tick protein was used
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for normalization (Pohl et al., 2008). The cycling parameters were as follows: 10 min at 95 °C
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followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, and
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extension at 72 °C for 20 s. To confirm primer specificity to produce a single amplification
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product, a melting curve analysis was performed using the default parameters of the instrument,
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and representative products from the RT-qPCR were electrophoresed. Primer efficiency was
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measured with 2-fold serially diluted cDNA, and for the transcription analyses, 300 ng of cDNA
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was added to each reaction. The relative expression ratio of ABC transporter genes was
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calculated according to the mathematical model described by Pfaffl (2001) using the Relative
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Expression Software Tool (REST-MCS©, version 2) (Pfaffl et al., 2002). Each analysis was
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conducted on biological triplicates.
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2.7. Statistical analyses
The statistical significance of the cytotoxicity and doubling-time assays was analyzed by
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two-way ANOVA and Bonferroni tests. The statistical significance of RT-qPCR results was
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analyzed by one-way ANOVA and Tukey’s tests. The results are expressed as the mean ± S.D. P
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values ≤ 0.05 were considered to be statistically significant.
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3. Results
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3.1. Selection of BME26-IVM cells
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Prior to the selection of IVM-resistant cells, the effect of IVM on the growth of the
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parental BME26 cells was analyzed (Fig. 1). BME26 cells were incubated at different
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concentrations of IVM over a 28-day period. No significant difference was observed in the
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doubling-time of non-IVM-exposed cells (13 ± 0.4 days), compared with that of cells treated
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with 0.5 µg/mL IVM (11 ± 2.5 days). BME26 cells treated with 2.5 µg/mL IVM grew relatively
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more slowly, with a significantly longer doubling-time (30 ± 0.3 days). When treated with higher
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IVM doses (12.5 or 62.5 µg/mL), the cells did not survive (Fig. 1). Cell viability was not
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affected by methanol (IVM solvent) (data not shown). Based on these data, an IVM-resistant cell
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line (BME26-IVM) was established after 46 weeks by exposing the parental BME26 cells to
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increasing IVM concentrations, starting at 0.5 µg/mL and ultimately reaching 6 µg/mL.
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3.2. Characterization of BME26-IVM cells The growth rates of the drug-sensitive parental cells (BME26) and the IVM-resistant cells
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(BME26-IVM) were compared. Both had the same doubling time, 16 ± 1 days, when grown in
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medium without IVM (Fig. 2A, - IVM). The BME26-IVM cells incubated with 6 µg/mL IVM
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had a doubling time of 12 ± 1 days, which was not significantly different from that of the cells
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incubated without IVM. However, the BME26 cells incubated with 6 µg/mL IVM had a much
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longer doubling time, 34 ± 4 days (Fig. 2A, + IVM), indicating that IVM had a cytotoxic effect
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on the drug-sensitive cells. Due to the cytotoxicity of IVM, the BME26 cells became more
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vacuolated (Fig. 2B) than the BME26-IVM cells, which retained a similar morphology to that of
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the untreated cells (Fig. 2B).
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3.3. IVM resistance determination and evaluation of involvement of ABC transporters in IVM
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resistance
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To evaluate IVM resistance, the viabilities of the BME26 and BME26-IVM cell lines
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were examined after exposure to different concentrations of IVM in the presence or absence of
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the ABC transporters inhibitor CsA (Fig. 3A). The cellular viability of BME26 cells was
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significantly reduced, compared with that of BME26-IVM cells after treatment with increasing
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IVM concentrations (Fig. 3A). The LC50 of BME26 cells (3.35 ± 0.09 µg/mL) was significantly
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lower than that of BME26-IVM cells (15.1 ± 1.6 µg/mL) (Fig. 3B), demonstrating that the
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BME26-IVM cells were 4.5 times more resistant to IVM than the BME26 cells. The cellular
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viability of BME26-IVM cells co-incubated with CsA was significantly reduced compared with
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that of BME26-IVM cells not exposed to CsA (Fig. 3A). The LC50 of the BME26-IVM cells co10 Page 10 of 26
incubated with CsA was significantly decreased compared with the the LC50 of the cells not
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exposed to CsA (15.10 ± 1.6 µg/mL and 8.42 ± 0.83 µg/mL, respectively), indicating a reduction
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in the resistance to IVM by a factor of 1.79 (Fig. 3B). In contrast, CsA had no effect on BME26
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cells (Fig. 3A and B).
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3.4. Expression of ABC transporters in IVM-resistant cells
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Because ABC transporters are involved in IVM resistance, the transcription levels of
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ABC transporters were investigated. The ABC transporters expression profiles (RmABCC1,
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RmABCC2, RmABCB7, and RmABCB10) were determined by RT-qPCR analysis of BME26 and
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BME26-IVM cells in the presence or absence of IVM. As shown in Fig. 4, RmABCB10 gene
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expression was significantly increased in BME26-IVM cells exposed to IVM compared with
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untreated cells. A significant increase in RmABCC1 expression was also observed in both
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BME26 and BME26-IVM cells treated with 9 µg/mL IVM. Whereas RmABCB7 was only
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upregulated in BME26 cells exposed to IVM, the expression levels of RmABCC2 were not
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significantly different across all treatments. These results demonstrate that ABC transporters are
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involved in the cellular response to IVM exposure.
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4. Discussion
In the present study, an IVM-resistant cell line was selected to elucidate the metabolic
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basis of IVM resistance in ticks. This cell line was obtained after exposing the embryonic cell
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line BME26 to increasing but non-lethal doses of IVM. The BME26-IVM cell line was 4.5 times
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more resistant than the parental BME26 cells, an RR similar to that observed in field tick
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populations resistant to IVM (Pohl et al., 2011).
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Cell lines derived from human cancer have been used to identify genetic alterations
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responsible for drug resistance (Gillet et al., 2007; Wind and Holen, 2011). In addition, previous
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studies have also shown that insect- and tick-derived cell lines are useful tools for evaluating the
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molecular mechanisms of drug resistance. Joussen et al. (2008) showed that the gene Cyp6g1, a
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cytochrome P450 monooxygenase, is overexpressed in cultured cells from Nicotiana tabacum,
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which metabolizes the insecticides DDT (dichlorodiphenyltrichloroethane) and imidacloprid.
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This same gene is overexpressed in Drosophila melanogaster strains resistant to a wide range of
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insecticides, including DDT and imidacloprid (Daborn et al., 2001). Similar results were
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observed for R. microplus, as both organophosphate-resistant cell lines (Cossio-Bayugar et al.,
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2002) and organophosphate-resistant tick populations showed increased levels of esterase
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activity (Rosario-Cruz et al., 1997). These reports showed that similar phenotypes are observed
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in vivo and in vitro, thus supporting the concept of the development of drug-resistant cell lines as
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a useful tool to study the mechanisms of cellular drug resistance.
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Over the past 30 years, in vitro studies have led to the identification of approximately 400
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genes whose expression levels affect the response to chemotherapy in human cancer cells (Gillet
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and Gottesman, 2010). Among those genes, the ABC transporters, a superfamily of 48 highly
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homologous members classified into seven subfamilies, play an important role in the export of
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drugs from cells, thereby affecting the bioavailability and effectiveness of the drugs (Szakács et
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al., 2006; Gillet et al., 2007).
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The expression levels of ABC transporters has also been associated with drug resistance
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in arthropods and nematodes. The overexpression of ABC transporters was associated with IVM
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resistance in Haemonchus contortus (Xu et al., 1998), Caenorhabditis elegans (James and
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Davey, 2009), Pediculus humanus humanus (Yoon et al., 2011), Sarcoptes scabiei (Mounsey et
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al., 2010), and R. microplus (Pohl et al., 2011). The influence of IVM on the expression of ABC
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transporters has also been investigated in cultured cells. Ménez et al. (2012) showed that IVM
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induced a time- and concentration-dependent upregulation of the mRNA levels of two ABC
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transporters in cultured mouse hepatocytes. Luo et al. (2013) also found that avermectins, 16-
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membered macrocyclic lactone derivatives that include IVM, induced an upregulation in protein
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level of an ABC transporter in D. melanogaster S2 cells.
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Corroborating these reports, our results showed that RmABCC1, RmABCB7, and
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RmABCB10 were overexpressed in cells treated with IVM. Furthermore, the incubation of
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BME26-IVM cells with a known ABC transporter inhibitor, CsA, partly reversed the resistance
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increasing the IVM toxicity. Taken together, these results suggest that ABC transporter proteins
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play a significant role in the development of resistance to IVM in this cell line. Nevertheless,
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because the reversal of resistance was only partial with the ABC transporter inhibitor, our results
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suggest that IVM resistance in R. microplus is multifactorial. The roles of detoxification
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enzymes, such as esterases, cytochrome P450 monooxygenases, and glutathione-S-transferases
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should also be considered, although these roles have not yet been shown with respect to IVM
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resistance in R. microplus.
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In a previous study, we reported that the inhibitor CsA increased IVM toxicity in resistant
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tick larvae and adults. The same ABC transporter gene that was shown to be upregulated in the
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present study (RmABCB10) has also been shown to be upregulated in an IVM-resistant tick
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population (Pohl et al., 2011).
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5. Conclusion
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Our results support the idea that similar biochemical mechanisms of IVM drug resistance
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are involved in vitro and in vivo, thus validating the use of the IVM-resistant BME26-IVM cell
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line to study IVM resistance development in the tick. Moreover, in vitro assays allow the
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experimental parameters to be rigorously controlled, thus reducing variability, which is often
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difficult to achieve in a bioassay. In conclusion, the establishment of acaricide-resistant cell line
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is useful for the study of the mechanisms of resistance to IVM and other drugs, which remain to
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be better characterized.
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Competing Interests
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The authors have declared that no competing interests exist.
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We thank CNPq - Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular,
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Acknowledgments
CAPES, FAPESP, FINEP and FAPERGS for supporting our work.
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Uchino, K., Tamura, T., Mita, K., Kadono-Okuda, K., Wada, S., Kanda, K., Goldsmith, M.R.,
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Figure Captions
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Fig. 1- BME26 cell growth in the presence of increasing ivermectin (IVM) concentrations. Cells
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were cultured without or with IVM at a concentration of 0.5, 2.5, 12.5 or 62.5 µg/mL. Viable
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cells were counted every 7 days over a 28-day culture period. All measurements are expressed as
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the mean ± S.D., n= 3. The asterisks (*) denote significant differences from the control (p≤ 0.05).
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Fig. 2- Effects of ivermectin (IVM) on cell growth. A- Cells were cultured in medium without (-
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IVM) or with (+ IVM) 6 µg/mL IVM. Viable cells were counted.every 7 days over a 28-day
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culture period. All measurements are expressed as the mean ± S.D., n= 3. The asterisks (*)
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denote significant differences (p≤ 0.05). B- Panoptico-stained BME26 and BME26-IVM cells
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after a 14-day incubation period in medium without (- IVM) or with (+ IVM) 6 µg/mL IVM, as
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observed by light microscopy (10x 1000x magnification).
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Fig. 3- Ivermectin (IVM) resistance determination and the effects of Cyclosporin A (CsA) on
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IVM toxicity. Cells were incubated in complete medium supplemented without or with IVM at a
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concentration of 1, 3, 6 or 9 µg/mL in the absence or presence of 5 µM CsA. After a 9-day
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incubation period, the cell viability was determined. A- The percentage of viable cells is
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expressed as the percentage of surviving cells in medium without IVM (100% cell viability). All
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measurements are expressed as the mean ± S.D., n= 3. The asterisks (*) denote significant
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differences from BME26-IVM (p ≤ 0.05). B- The LC50 values for BME26 and BME26-IVM
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cells in the presence or absence of the ABC transporter inhibitor CsA. The different
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superscripted letters following the values indicate statistical significance (p ≤ 0.05).
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was estimated by dividing the LC50 of the BME26-IVM cells by that of the parental BME26
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cells. B The fold-reversal factor was estimated by dividing the LC50 of the cell line treated with
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IVM and without CsA by that of the cell line treated with both IVM and CsA.
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The RR
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Fig. 4- Relative transcription levels of ABC transporter genes in BME26 and BME26-IVM cells
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cultured in medium without IVM or medium with 6 µg/mL or 9 µg/mL IVM. After a 9-day
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incubation period, RNA was extracted from these cells, and ABC transporters mRNA was
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analyzed by RT-qPCR. The data represent the mean ± S.D. of three independent experiments,
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normalized to 40-S transcript levels. The letter a denotes significant differences (p≤ 0.05) from
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BME26 cells (- IVM), and b denotes significant differences (p ≤ 0.05) compared with all
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treatments.
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Table 1- Primers used in the relative quantification of R. microplus ABC transporter mRNAs by
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RT-qPCR. Amplicon
NCBI Primer
Gene
Sequence 5’-3’
Tm size (bp)
EW679928
RmABCC1
JN098447
RmABCB10
TGA GGC GAA CCT GGT TGT GCT GAG CG
Forward
GAC ACC ATT CAC CGA GAG TTC AGT AGC AC
61.9
Reverse
GCC CTG CTC CAC TAT TTC GCC ACC
64.2
Forward
CGC GGG ACC TTC TGA AGC
58.9
Reverse
GGT AGC TCG GTA TAG GGC TAG ACG
59.8
Forward
AGTGATGGCAGAGTGTGTGAACGG
59.9
Reverse
CATTCTCCTGGTGGGCTTGATGC
61.4
Forward
GCC GCA GTT GTC ACT TGT TGG TTT G
61.3
Reverse
ACG TCC GCT GCC ACT TGC CTC
JN098448
an
RmABCB7
Reverse
JX170903
JN098446
M
RmABCC2
ACG ACC GAT GGC TAC CTC CTC CGC
106
59.2
120
84
109
95
64.9
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452 453
59.1
cr
ribosomal
Forward
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40S
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accession no.
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