Transcriptional responses in Parascaris univalens after in vitro exposure to ivermectin, pyrantel citrate and thiabendazole

Abstract

Background: Parascaris univalens is a pathogenic parasite of foals and yearlings worldwide. In recent years Parascaris spp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development is unknown. The aim of this study was to investigate the transcriptional responses in adult P. univalens worms after in vitro exposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways. Methods: Adult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10-9 M, 10-11 M, 10-13 M), pyrantel citrate (10-6 M, 10-8 M, 10-10 M), thiabendazole (10-5 M, 10-7 M, 10-9 M) or without anthelmintics (control) at 37 °C for 24 h. After incubation the viability of the worms was assessed and RNA extracted from the anterior end of 36 worms and sequenced on an Illumina NovaSeq 6000 system. Results: All worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (p < 0.05 and fold change ≥ 2) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes up- or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP) as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be up- and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunit lgc-37, which was upregulated only in worms exposed to 10-9 M of ivermectin. Conclusions: This is the first time the expression of these genes has been described in P. univalens and future work should be focused on characterising these candidate genes further to explore their potential involvement in drug metabolism and anthelmintic resistance.

Ivermectin-induced Gene Expression Changes in Adult Parascaris Univalens and Caenorhabditis Elegans- a Comparative Approach to Study Anthelminthic Metabolism and Resistance in Vitro

“…Expression of the Phase II enzyme, ugt-54, was decreased in P. univalens after IVM exposure, but no response was observed in C. elegans. Similarly, decreased expression of other members of the UGT family, ugt-3, ugt3a1, ugt-47 and ugt-48, have been observed in P. univalens in vitro exposed to pyrantel citrate, thiabendazole and oxibendazole (29,30). Although we did not observe any response of ugt-54 in C. elegans, other studies have shown differential gene expression of UGTs in IVM-tolerant strain of C .elegans after exposure to 10 −6 M IVM (74) and in the BZ-resistant strain of C. elegans after exposure to BZs (73,75,76).…”

“…For example, due to the di culties in obtaining live adult P. univalens, only three individuals were used per biological replicate in the study, compared to 300 worms for C. elegans. In addition, the unnatural in vitro culture environment may in uence undesired behavioral changes or gene expression in P. univalens worms (29). Furthermore, only 1.4% of RNAseq-derived DEGs from P. univalens were used as candidate genes in this study, of which some transcripts (splice variants) also showed varying expression patterns.…”

“…Adult P. univalens worms collected from two anthelmintic-naïve Icelandic foals at an abattoir in Selfoss, Iceland were exposed to IVM in vitro. A detailed account of the experimental setup has been described in Martin et al (29). Brie y, worms were incubated for 24 h in cell culture media containing 0.1% DMSO supplemented with 10 − 13 , 10 − 11 or 10 − 9 M IVM.…”

Drug efficacy on zoonotic nematodes of the Anisakidae family: new metabolic data

“…(2001). Larvae of C. osculatum (s. s.) (320 individuals) and P. decipiens (s. s.) (288 individuals) were cultured in vitro for 12 or 72 h, respectively, with IVM or PYR [dissolved in 0.1% dimethylsulphoxide (DMSO); 45684, Sigma Aldrich, Poznan, Poland] at three concentrations: 1.56 (lowest), 3.125 (medium) and 6.25 (highest) μ g mL −1 of culture medium (Hu et al ., 2013; Jones et al ., 2015; Martin et al ., 2020) (File S1.2). The control culture did not contain any of the tested anthelmintics (the medium had an addition of 0.1% DMSO).…”

Comparative Analysis on Transcriptomics of Ivermectin Resistant and Sensitive Strains of Haemonchus Contortus

“…Fortunately, the high-quality reference genome of this parasite together with the increasing accessibility to high throughput RNA sequencing offers an opportunity to facilitate aimed studies to explore its resistant mechanism. Transcriptome sequencing has been used widely to identify candidate genes/pathways related to drug resistance [17,18]. However, the transcriptome changes of H. contortus IVM eld resistant strain induced by natural selection, have received little attention.…”