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ATP-binding cassette (ABC) transporters are responsible for pumping drugs across membranes and are an important drug detoxification mechanism. Since ABC transporters act on a wide spectrum of chemical compounds, they have been associated with multidrug resistance phenotype in various parasites and cancer cells. Here, we document the presence of a Rhipicephalus (Boophilus) microplus tick population (Jaguar) resistant to four acaricide classes (organophosphates (OP), synthetic pyrethroids (SP), amitraz and macrocyclic lactones (ML)) and reveal that the cattle tick has a multidrug detoxification mechanism based on ABC transporter proteins. Acaricide toxicity was assessed using the larval packet test (LPT), and mortality data were subjected to probit analysis using a susceptible strain (POA) as reference. Larvae were pre-exposed to sub-lethal doses of the ABC-transporter inhibitors, cyclosporin A (CsA) and MK571, and subsequently treated with ivermectin, abamectin, moxidectin, chlorpyriphos, cypermethrin, or amitraz in LPT. Results show that lethal concentrations 50 % (LC(50)) of ivermectin, abamectin, moxidectin (MLs), and chlorpyriphos (OP) were significantly reduced in larvae exposed to CsA and MK571 inhibitors in the Jaguar resistant population, but LC(50) did not change in POA susceptible strain larvae. LC(50) of cypermetrin (SP) and amitraz remained unchanged in inhibitor-exposed larvae, compared to larvae from Jaguar and POA strains not exposed to inhibitor. These results suggest that ABC transporter proteins can protect ticks against a wide range of acaricides and have an important implication in drug resistance development as a multidrug detoxification mechanism.

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.

The tick Rhipicephalus (Boophilus) microplus (formerly Boophilus microplus) is the major ectoparasite affecting livestock in America, Asia, Africa, and Oceania. Conventional tick control is based on the use of acaricides but immunization of bovines with tick gut proteins induces only a partial protective immune response. Based on this information, distinct research groups have explored the possibility of protecting the animals by inducing an immune response against other tick proteins. However, the antigens so far described do not induce the necessary protection for suppressing the use of acaricides. Currently, several groups are engaged in identifying new tick proteins to be used as targets for the development of new vaccines. This approach focuses on the enhancement of the immunogenicity of antigens already tested by incorporating new adjuvants or formulations and by searching for new antigens. This paper reviews the work done by Brazilian researchers to develop a vaccine against this tick.Keywords: Boophilus microplus, Rhipicephalus microplus, vaccine, tick, control. ResumoO carrapato Rhipicephalus (Boophilus) microplus (anteriormente Boophilus microplus) é o principal ectoparasita que afeta bovinos na América, Ásia, África e Oceania e o seu controle é tradicionalmente realizado através do uso de acaricidas. Experimentos de imunização com proteínas do carrapato mostram que a resposta imune desenvolvida pelos bovinos vacinados protege, em parte, os animais do parasitismo. Baseado nessas observações, vários grupos de pesquisa exploram a possibilidade de proteger os animais pela indução de uma resposta imune contra proteínas do carrapato. Entretanto, os antígenos já caracterizados não asseguram o grau de proteção necessário para suprimir o uso de acaricidas. Portanto, esses grupos de pesquisa estão engajados na tentativa de identificar novas proteínas que possam ser utilizadas para o desenvolvimento de novas vacinas, as quais possam induzir maior imunogenicidade de que os antígenos já testados, através do uso de novas formulações e/ou pela incorporação de adjuvantes. O presente artigo apresenta uma revisão da literatura sobre os resultados obtidos por pesquisadores brasileiros no desenvolvimento de vacinas contra o carrapato.

In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new molecular mechanism of resistance to pesticides.

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