In vitro establishment of ivermectin-resistant Rhipicephalus microplus cell line and the contribution of ABC transporters on the resistance mechanism

Abstract

The cattle tick Rhipicephalus microplus is one of the most economically damaging livestock ectoparasites, and its widespread resistance to acaricides is a considerable challenge to its control. In this scenario, the establishment of resistant cell lines is a useful approach to understand the mechanisms involved in the development of acaricide resistance, to identify drug resistance markers, and to develop new acaricides. This study describes the establishment of an ivermectin (IVM)-resistant R. microplus embryonic cell line, BME26-IVM. The resistant cells were obtained after the exposure of IVM-sensitive BME26 cells to increasing doses of IVM in a step-wise manner, starting from an initial non-toxic concentration of 0.5 μg/mL IVM, and reaching 6 μg/mL IVM after a 46-week period. BME26-IVM cell line was 4.5 times more resistant to IVM than the parental BME26 cell line (lethal concentration 50 (LC50) 15.1 ± 1.6 μg/mL and 3.35 ± 0.09 μg/mL, respectively). As an effort to determine the molecular mechanisms governing resistance, the contribution of ATP-binding cassette (ABC) transporter was investigated. Increased expression levels of ABC transporter genes were found in IVM-treated cells, and resistance to IVM was significantly reduced by co-incubation with 5 μM cyclosporine A (CsA), an ABC transporter inhibitor, suggesting the involvement of these proteins in IVM-resistance. These results are similar to those already described in IVM-resistant tick populations, and suggest that similar resistance mechanisms are involved in vitro and in vivo. They reinforce the hypothesis that ABC transporters are involved in IVM resistance and support the use of BME26-IVM as an in vitro approach to study acaricide resistance mechanisms.

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

“…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 ., , , ). 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.…”

Tick Cell Lines in Research on Tick Control

“…The authors showed that ABC pump expression was not significantly modulated by ivermectin treatment, and expression of one of the subfamily genes was not detected. Interestingly, this study revealed that the IRE/CTVM19 cell line was able to tolerate a much higher concentration of ivermectin (30 µg/ml) than the R. microplus-derived line BME26 that did not survive after exposure to 12.5 µg/ml ivermectin (Pohl et al, 2014). This could have been due to biological differences between the two tick species from which the cell lines were derived, differences in the phenotypic composition of the cell lines and/or in the culture conditions used.…”

“…For some of the currently available R. microplus cell lines, the acaricide resistance status of the parent ticks from which the cultures were derived is known. As already mentioned, the parent BmVIII-SCC and BME26 cells used by, respectively, Cossio- Bayugar et al (2002b) and Pohl et al (2014) to derive acaricide-resistant sub-lines in vitro were originally susceptible to the compounds used, implying that the parent ticks of both lines, originally from Mexico, were also acaricide-susceptible. Two additional R. microplus cell lines, BME/CTVM2 (Bell-Sakyi, 2004) and BME/CTVM4 (Bell-Sakyi et al, 2007), were derived from known acaricide-susceptible ticks from Costa Rica, while two other cell lines, BME/CTVM5 (Bell-Sakyi et al, 2007) and BME/CTVM6 (Bell-Sakyi, 2004) were derived from Colombian ticks known to be resistant to organophosphates, organochlorines and amitraz (Koh-Tan et al, 2016).…”

“…A study conducted by Pohl et al (2014) also focused on understanding the mechanisms of acaricide resistance in R. microplus. In this study, the authors established an ivermectin-resistant sub-line (BME26-IVM) of the R. microplus embryo-derived cell line BME26 (Kurtti et al, 1988;Esteves et al, 2008) by exposing susceptible cells to increasing concentrations of ivermectin (Table 2) over 46 weeks.…”

Characteristics and application of tick cell lines

“…The forefront of medical entomology and approaches towards tick-borne diseases control 2 Chen et al, 1994Munderloh et al, 1994 28 34°C The data are retrieved from the website of the Cellosaurus (https://web.expasy.org/cellosaurus/) as of July 2019. Sterba et al, 2014;Tonk et al, 2014a;Tonk et al, 2014bSchnettler et al, 2014Rosa et al, 2016;Smith et al, 2016Cramaro et al, 2017Miller et al, 2018Pohl et al, 2014Koh-Tan et al, 2016;Mangia et al, 2016Oltean et al, 2013Antunes et al, 2014Contreras et al, 2017Yoshii et al, 2008Bell-Sakyi et al, 2012;Mazelier et al, 2016Blouin et al, 2002Socolovschi et al, 2009;…”