Ascarids exposed: a method for in vitro drug exposure and gene expression analysis of anthelmintic naïve Parascaris spp

Abstract

Ascarid parasites infect a variety of hosts and regular anthelmintic treatment is recommended for all species. Parascaris spp. is the only ascarid species with widespread anthelmintic resistance, which allows for the study of resistance mechanisms. The purpose of this study was to establish an in vitro drug exposure protocol for adult anthelmintic-naïve Parascaris spp. and report a preliminary transcriptomic analysis in response to drug exposure. Live worms were harvested from foal necropsies and maintained in RPMI-1640 at 37 °C. Serial dilutions of oxibendazole (OBZ) and ivermectin (IVM) were prepared for in vitro drug exposure, and worm viability was monitored over time. In a second drug trial, worms were used for transcriptomic analysis. The final drug concentrations employed were OBZ at 40.1 μm (10 μg mL-1) and IVM at 1.1 μm (1 μg mL-1) for 24 and 3 h, respectively. The RNA-seq analysis revealed numerous differentially expressed genes, with some being potentially related to drug detoxification and regulatory mechanisms. This report provides a method for in vitro drug exposure and the phenotypic responses for Parascaris spp., which could be extrapolated to other ascarid parasites. Finally, it also provides preliminary transcriptomic data following drug exposure as a reference point for future studies of Parascaris spp.

Keywords: Anthelmintic; Parascaris; ascarid; in vitro; transcriptome.

Fig. 1.

An illustration of the study design (IVM, ivermectin; OBZ, oxibendazole).

Fig. 2.

A graphical representation of mean worm viability following in vitro anthelmintic exposure. Control worms were maintained in RPMI 1640 medium only or with dimethyl sulfoxide (DMSO) which was used to prepare the anthelmintics. Error bars represent 95% confidence intervals (α = 0.05).

Fig. 3.

A graphical representation of mean worm viability of worms used in Part 2 of this study, where those exposed to oxibendazole (OBZ) at 40.1 μm (10 μg mL⁻¹) for 24 h and ivermectin (IVM) at 1.1 μm (1 μg mL⁻¹) for 3 h were used for RNA-sequencing analysis. Control worms were maintained in RPMI 1640 medium only or with dimethyl sulfoxide (DMSO) which was used to prepare the anthelmintics. Error bars represent 95% confidence intervals (α = 0.05).

Fig. 4.

A graphical representation of select genes from the RNA-seq analysis performed in Part 2 of this study. The housekeeping genes are ama and ncbp. (A) In vitro vs in situ controls, (B) all control worms vs all treated worms, where the selected genes had significantly higher expression in the treated worms (α = 0.01), (C) ivermectin (IVM) treated with 1.1 μm (1 μg mL⁻¹), IVM control, oxibendazole (OBZ) treated with 40.1 μm (10 μg mL⁻¹), and OBZ control.

Fig. 5.

A graphical representation of the gene ontology pathway analysis for significantly different genes (SDGs) between groups. From left to right: all genes, all treated worms vs all control worms, ivermectin (IVM) treated with 1.1 μm (1 μg mL⁻¹) vs IVM control, oxibendazole (OBZ) treated with 40.1 μm (10 μg mL⁻¹) vs OBZ control. The top row reflects biological processes (BP) and the bottom row reflects molecular functions (MF).