For several parasites of veterinary importance there is a

For several parasites of veterinary importance, there is a growing body of evidence that resistance to ML is, at least in part, mediated by ABC transporters (P-glycoproteins) (Whittaker et al., 2017). P-glycoproteins (Pgps) are members of the ATP-binding cassette transporter family that are well known to modulate drug resistance in bacteria and neoplastic tissues (Davidson and Chen, 2004; Licht et al., 1994). In helminths, Pgps are thought to contribute to drug resistance by effluxing anthelmintics away from their molecular target. Pgps associated with ML resistance have been characterized in several nematode parasites including the trichostrongyle Haemonchus contortus (Godoy et al., 2015a, 2016; Godoy et al., 2015b; Xu et al., 1998), cyathostomins (Drogemuller et al., 2004; Kaschny et al., 2015), and the filarid Dirofilaria immitis (Bourguinat et al., 2016; Mani et al., 2016). Recent evidence suggests that increased levels of Pgp expression are highly correlated with reduced susceptibility of Parascaris to MLs (Janssen et al., 2013). Two Pgp genes, Peq-Pgp-11 and Peq-pgp-16, have been identified. Of these two Pgp genes, it appears that Peq-pgp-11 is more strongly associated with resistance (Janssen et al., 2013). Decreased susceptibility to MLs has been demonstrated with transgenically expressed Peq-pgp-11 in C. elegans (Janssen et al., 2015). It is thought that the ML resistant phenotype occurs when expression of Peq-pgp-11 is high, however, the factors regulating this expression are not well characterized (Nielsen et al., 2014). Pgp-11 orthologs that alter susceptibility to MLs have been shown to occur in other parasitic nematodes including D. immitis (Mani et al., 2016), Cooperia oncophora (De Graef et al., 2013), H. contortus (Raza et al., 2016) and non-parasitic Caenorhabditis elegans (Bygarski et al., 2014). Pgp-16 orthologs occur in H. contortus (Godoy et al., 2015a) and in C. oncophora (Demeler et al., 2013; Tydén et al., 2014) with expression levels increased after ML exposure in the latter. In cp-690550 Pgp-16 orthologs were not found in C. elegans (Issouf et al., 2014). In Parascaris, tissue-specific expression of Pgp has been investigated by dissecting worm tissues and amplifying expressed genes by PCR (Janssen et al., 2013). However, this preparation method precludes making clean preparations of tortuous linear organs such as the testes (Janssen et al., 2013), and makes studying localization in nerve cords, and lateral cords impossible due to their small size. To overcome these obstacles, expression of Pgps can be studied in situ in sections of worm tissue that allow the preservation of anatomy and tissue architecture. We examined Pgp expression using an in situ multiple nucleic acid RNA hybridization method that allowed visualization of individual mRNA transcripts through a novel signal amplification technique (Wang et al., 2012). This method utilizes a double Z probe design based on a target mRNA with the base of the Z having a complementary binding sequence. The 18- to 25- nucleotide base and the 14- nucleotide tail of the Z are connected by a spacer. Binding of two adjacent Z probes allows the formation of a 28 nucleotide binding site made of two tails of the Z probes. A preamplifier binds to the site, and is turn bound by an amplifier, which is bound by an alkaline phosphatase labeled signal amplifying probe. A chromogenic substrate is added that allows visualization. The chromogenic signal, indicating an individual mRNA transcript, can be viewed as a punctate dot by light microscopy. The aim of this present study was to determine the tissue distribution of Peq-pgp-11 and Peq-pgp-16 as determined by multiple nucleic acid RNA hybridization.
Materials and methods
Results
Discussion P-glycoproteins have been associated with macrocyclic lactone resistance in nematode parasites, including Parascaris. This is the first study to examine in situ P-glycoprotein mRNA localization in an ascarid or equine nematode parasite. More broadly, our results suggest the multiple nucleic acid hybridization technique can be used as a quantitative measure of mRNA transcripts in parasitic nematodes.