Exposure to amitraz, fipronil and permethrin affects cell viability and ABC transporter gene expression in an Ixodes ricinus cell line

Abstract

BackgroundOver-expression of ATP-binding cassette (ABC) transporter proteins has been implicated in resistance of ticks to acaricides. Tick cell lines are useful for investigating resistance mechanisms, as development of an in vitro model for the study of acaricide resistance would contribute to improving knowledge of the molecular basis behind drug processing and exclusion in ticks. In the present study, cultures of the Ixodes ricinus-derived cell line IRE/CTVM19 were treated with the acaricides amitraz, permethrin or fipronil to determine modulation of ABC transporter gene expression. Cells were treated with different drug concentrations (25, 50, 100, 150 μM) and incubated for ten days. Cell morphology, viability, metabolic activity and relative expression of ABC (B1, B6, B8 and B10) genes were determined at day 10 post-treatment.ResultsCell morphology determined by light microscopy was altered following treatment with all drugs, but only at high concentrations, while total cell numbers decreased with increasing drug dose. Cell viability determined by trypan blue exclusion was not significantly different from untreated controls (P > 0.1) following treatment with amitraz and permethrin, but high concentrations of fipronil caused decrease (up to 37%, P < 0.01) in viability. At all drug concentrations, fipronil and permethrin induced dose-dependent reduction in cell metabolic activity measured by MTT assay (P < 0.01). Quantitative RT-PCR showed that the drugs significantly affected expression of ABC genes. In particular, fipronil treatment downregulated ABCB1 (P < 0.001) and upregulated ABCB6, ABCB8 and ABCB10 (P < 0.01); amitraz treatment down regulated ABCB1 (significant difference between 25 and 150 μM, P < 0.001) and upregulated ABCB8 and ABCB10 at lower concentrations (25 and 50 μM, P < 0.05); and permethrin upregulated ABCB6, ABCB8 and ABCB10 only at 150 μM (P < 0.01).ConclusionsThe adverse effects on cell viability and metabolic activity, and changes in expression of different ABC transporter genes, detected in IRE/CTVM19 cells following treatment with amitraz, permethrin and fipronil, support the proposed application of tick cell lines as in vitro models for the study of resistance to these acaricides in ticks.

Pyriproxyfen, a juvenile hormone analog, damages midgut cells and interferes with behaviors of Aedes aegypti larvae

“…Midgut of treated larvae has damaged epithelial layer, with cell debris released in the midgut lumen, similar to found in this insect exposed to other new chemistry insecticides (Gaban et al, 2015). Cytoplasm vacuolization followed by cell breakdown releasing cell debris into the midgut lumen has been associated with digestive cell death caused by xenobiotics in different insects (Costa et al, 2014; Fiaz et al, 2018a; Suresh Kumar et al, 2013; Mangia et al, 2018; Martínez et al, 2018b).…”

Genetic analysis and screening of detoxification-related genes in an amitraz-resistant strain of Panonychus citri

“…ELF1α and ACTB genes from the citrus red mites were used as internal reference genes (Niu et al, 2012). Primers for the DEGs and internal reference genes were designed using Primer Premier 6 software (table S1).…”

“…The results of differentially expressed genes (DEGs) and modeled KEGG pathway in amitraz-treated Rhipicephalus decoloratus laid the basis for further investigation on resistance mechanisms (Baron et al, 2018). The stability of cell line from Ixodes ricinus was affected after treatment with amitraz, and ABC transports of cell line were upregulated or downregulated to varying degrees, indicating that this cell line was fit for in vitro mechanistic studies of amitraz resistance (Mangia et al, 2018). Functional analyses through in vitro expression of β-adrenergic-like octopamine receptor in Plutella xylostella, Huang et al found that amitraz, octopamine, and tyramine all activate the β-adrenergic-like octopamine receptor, leading to elevated cyclic AMP levels.…”

Tick Cell Lines in Research on Tick Control

“…This should be considered when conducting such in vitro experiments using tick cell lines. In a further study, Mangia et al (2018) treated IRE/CTVM19 cultures with different concentrations of the acaricides permethrin, amitraz and fipronil ( Table 2). They assessed cell viability, morphology, metabolic activity and expression of ABC genes at day 10 post-treatment.…”