Polymorphisms in exon 11 of the mptl-1 gene and monepantel resistance in Haemonchus contortus

Chemical treatments are the main strategy to control gastrointestinal nematodes in sheep, and the emergence of anthelmintic resistance, as consequence, results in control failures and leads to economic losses. Thus, molecular tests may constitute an excellent tool for the early detection of anthelmintic resistance-related mutations. Thus, a polymerase chain reaction (PCR)-based genotyping assay followed by polyacrylamide gel electrophoresis (PAGE) was developed to detect polymorphisms in exon 11 of the acetylcholine receptor monepantel-1 gene (mptl-1) that were previously associated with monepantel resistance through a genome-wide study in Haemonchus contortus. DNA samples recovered from individual and pooled third-stage larvae from two susceptible field-derived isolates and five (three in vivo-derived and two field-derived) resistant populations were used. New polymorphisms, including a 6-bp deletion and a 3-bp insertion, were detected in resistant individuals. These indels, confirmed using sequencing of cloned PCR products, are predicted to result in amino acid changes in transmembrane domain 2 (TMD2) of the MPTL-1 protein. The two susceptible isolates showed only the presence of the wild-type allele (100%), whereas lower frequencies of the wild-type allele were detected in monepantel-resistant populations (11.1 to 66.7%). These findings report new polymorphisms in the mptl-1 gene, validate the results obtained through genomic mapping for monepantel resistance, and provide a PCR-based assay to genotype indels located in exon 11 of mptl-1 in H. contortus.


Introduction
Gastrointestinal nematode infections, especially those caused by H. contortus (the barber's pole worm), are responsible for large economic losses in sheep production owing to decreased performance, treatment expenses, and anthelminthic resistance (Miller et al. 2012;Chagas et al. 2022). As anthelmintic resistance has become a worldwide problem, tests for resistance using molecular markers are an attractive alternative for the identification of early resistance emergence, supporting decisions on treatment management (Gasser et al. 2008). Furthermore, screening tools based on molecular markers can guide the rational use of anthelmintics in flocks to improve monitoring of resistance in the field. Thus, the widespread presence of resistance alleles can be avoided (Roos et al. 2004).
Considering the anthelmintic classes available for sheep treatment, molecular markers of resistance, as reviewed by Kotze et al. (2020), are only well-established for benzimidazoles (mutations in isotype 1 of the β-tubulin gene).
However, molecular markers for levamisole (mutations in the acr-8 gene) and amino-acetonitrile derivatives (AAD) (mutations in the mptl-1 gene) have only been partially elucidated (Kotze et al. 2020). In addition, molecular marker data are still inconsistent across studies of macrocyclic lactones in H. contortus; however, there is genomic evidence that a single major locus on chromosome V is associated with ivermectin resistance (Doyle et al. 2019). Genotyping assays based on sequencing or polymerase chain reaction (PCR) can be used to detect polymorphisms associated with anthelmintic resistance; the method of choice depends on the scale and resolution required (Kotze et al. 2020).
Monepantel, an AAD, is one of the most recently developed anthelmintic compound suitable for the control of sheep gastrointestinal nematodes that exhibited multiple resistance to previously introduced drug classes (Kaminsky et al. 2008(Kaminsky et al. , 2011. However, several years after its launch, monepantel resistance was reported in multiple flocks worldwide (Mederos et al. 2014;Van den Brom et al. 2015;Sales and Love 2016;Albuquerque et al. 2017). In H. contortus, monepantel acts on the acetylcholine receptor DEG-3-like protein (DEG-3) group of nematode-specific nicotinic acetylcholine receptors (nAChR), which comprise two receptors: one containing DEG-3 and the acetylcholine receptor DES-2-like protein (DES-2), and one containing MPTL-1 (reviewed by Holden-Dye et al. 2013). Monepantel and its metabolite, monepantel sulfone, are superagonists that open the MPTL-1 channel in an irreversible manner (Baur et al. 2015). As MPTL-1 is the main monepantel target receptor, several molecular changes in the mptl-1 gene, presumably leading to a non-functional protein, have been associated with monepantel resistance in H. contortus (Kaminsky et al. 2008;Rufener et al. 2009;Bagnall et al. 2017). In a previous extreme-quantitative trait loci (X-QTL) genomic mapping study, a selection sign on chromosome 2 in a region containing the mptl-1 gene was detected (Niciura et al. 2019). By comparing the allele frequency differences between samples collected before and after monepantel treatment, polymorphisms, mainly deletions, in exon 11 have been identified as potential causal mutations responsible for monepantel resistance (Niciura et al. 2019). Considering the large number and constant emergence of new polymorphisms reported in mptl-1, any assay for identification of polymorphisms related to resistance should address all possible mutations across the gene (Kotze et al. 2020) or, alternatively, fine-map regions identified by genome-wide approaches or investigate loci frequently affected in various populations. Therefore, to validate previous results obtained using genomic mapping for monepantel resistance in H. contortus, the present study examined polymorphisms in exon 11 of mptl-1 and designed a PCR-based assay for genotyping the identified indels.

H. contortus strains
Seven H. contortus populations previously known for their monepantel resistance status were used. Two monepantelsusceptible field-derived (SFD) isolates, retrieved before the monepantel launch on the Brazilian market, were named SFD1 and SFD2. SFD1, recovered in 2010, is susceptible to monepantel but resistant to other drugs (Chagas et al. 2013), and was the parental susceptible isolate used for parasite crossing in an X-QTL study for monepantel resistance (Niciura et al. 2019), whereas SFD2, recovered in 1990, is susceptible to several drugs (Echevarria et al. 1991). Three resistant isolates, obtained in 2018-2019 through in vivo selection using monepantel subdosing administration (monepantel doses from 0.075 to 2.5 mg/kg in 19-26 rounds of selection at minimum 14-day intervals for 112-133 weeks) in three different sheep hosts after artificial infection with third-stage larvae (L 3 ) from the SFD1 isolate (Niciura et al. 2020), were referred to as RIV1, RIV2, and RIV3. The two resistant fieldderived populations were named RFD1 and RFD2. RFD1 is a resistant isolate recovered in 2017 (Albuquerque et al. 2017) used as the resistant parent in the X-QTL for monepantel resistance (Niciura et al. 2019), and RFD2 is a population comprising 95.7% Haemonchus spp. and 4.3% Trichostrongylus spp. retrieved in 2022 from the Embrapa Pecuaria Sudeste sheep flock, in which persistently high egg counts were observed despite treatment with 2.5 mg/kg therapeutic monepantel dose. After collection, H. contortus isolates were cryopreserved and maintained under nitrogen at − 196 °C until passage through sheep hosts for larvae recovery. Pools and individual larvae from the six H. contortus isolates (SFD1, SFD2, RIV1, RIV2, RIV3, and RFD1) were used to investigate new polymorphisms in exon 11 of mptl-1. Additionally, individual larvae, using a larger sample size from the RFD2 population for validation, were used to assess the allelic and genotypic frequencies of polymorphisms.

DNA extraction
The DNA of individual and pooled (1,000 individuals) H. contortus L 3 was extracted using an organic solvent and diluted in water (Niciura et al. 2012). Briefly, larvae were exsheathed with 0.15% sodium hypochlorite and incubated in digestion buffer with 0.4 mg/mL proteinase K at 56 °C overnight. DNA was then extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and washed with 100% isopropanol and 70% ethanol. The protocols used for individual and pooled L 3 differed only in reagent volume and final dilution in water (10 µL for individual and 50 µL for pooled L 3 ). For pooled L 3 , DNA concentration and purity were estimated using ultraviolet absorbance (NanoDrop™ 2000, Thermo Fisher). Because very low yields were obtained for individual L 3 (10 µL per sample), the DNA concentration was not measured.
PCR products were subjected to both agarose and polyacrylamide gel electrophoresis. An adapted electrophoresis protocol using a 4% agarose gel (Bhattacharya and Van Meir 2019) with 1 × TBE at 75 V for 130 min and ethidium bromide staining (0.5 µg/mL in gel followed by 2.0 µg/mL for 20 min in a bath) was used. In addition, denaturing 6% polyacrylamide gel electrophoresis (PAGE) with 1 × TBE at 60 W for 2 h and silver staining was performed.
The susceptible homozygous genotype (named SS) presented two identical wild-type alleles, the resistant homozygous genotype (named RR) presented two copies of a mutated allele, and the heterozygous genotype (named RS) presented one wild-type allele and one mutated allele.

Cloning and Sanger sequencing
Pooled L 3 samples from each H. contortus isolate were amplified, cloned, and subjected to Sanger sequencing (5 or 6 clones per isolate). For amplification, a High-Fidelity Taq DNA Polymerase was used, and the PCR previously described was modified as follows: 0.2 µM of each primer and 1 × of PCRBio Ultra Mix (PCR Biosystems) in a final volume of 25 µL. PCR amplification was confirmed using 1.5% agarose gel electrophoresis.
For cloning, PCR products were purified using Wizard® SV Gel and the PCR Clean-Up System (Promega), and subjected to A-tailing, ligation, and transformation procedures using pGEM®-T Easy Vector Systems (Promega). White colonies were picked, subjected to clone insert verification using colony PCR (Green and Sambrook 2019) with amplicon-specific primers, and visualized on a 1.5% agarose gel. Plasmid minipreps from recombinant colonies were generated using the PureYield™ Plasmid Miniprep System (Promega), and plasmids were quantified using a Qubit™ dsDNA HS assay (Thermo Fisher) in a Qubit fluorometer (Thermo Fisher).
Plasmids were subjected to Sanger sequencing using M13 forward and reverse primers in two separate runs and a Big-Dye Terminator v3.1 cycle sequencing kit (Thermo Fisher), followed by analysis using an ABI Prism 3730XL DNA analyzer (Thermo Fisher). All nucleotide sequence data for full-length exon 11 of mptl-1 in H. contortus are available in GenBank (accession numbers: ON014540 to ON014573) (Supplementary Table 1). The resulting sequences from the forward and reverse runs were aligned using Bioedit to obtain contig sequences that were compared to the reference sequence (HCON_00039360) deposited on WormBase ParaSite (version WBPS16) and translated using Emboss Transeq and Emboss Sixpack software (Madeira et al. 2019).

Results
Initially, DNA from pooled L 3 of the six H. contortus isolates was subjected to PCR to amplify exon 11 of mptl-1, and different fragment sizes were observed after agarose gel electrophoresis. As further confirmed through sequencing, only the 177bp wild-type fragment was amplified in the SFD1 and SFD2 susceptible isolates, whereas products of 171, 177, and 180 bp were observed for RIV1-, RIV2-, RIV3-, and RFD1-resistant isolates (Fig. 1A). An additional band larger than 180 bp was observed for RFD1 (Fig. 1A), but it was not retrieved from samples submitted for cloning and sequencing. This additional band was excised from agarose gels, purified, and subjected to Sanger sequencing, but no contig sequence could be generated due to the 6 bp-mutated allele presence in samples, leading to electropherogram peak superposition after sequencing.
In addition to the indels, several SNPs were detected in exon 11 of mptl-1 in both resistant and susceptible isolates (Fig. 2). However, the designed PCR-based assay followed by electrophoresis is not suitable for detecting these point mutations.
The association between the indels detected in exon 11 of mptl-1 and monepantel resistance was addressed through PCR-based genotyping using individual H. contortus L 3 from an independent resistant field-derived population RFD2. Plasmidial DNA samples were used as positive controls for 171 bp (RFD1-clone 4), 177 bp (SFD2-clone 4), and 180 bp (RIV2-clone 4) fragment sizes. While 4% agarose gel electrophoresis was able to detect polymorphic banding patterns when a few samples were subjected to electrophoresis on small (6 cm × 12 cm) gels (Fig. 1A), curving of the edges of the gel, leading to wrong genotype attribution, occurred when larger (15 cm × 25 cm) agarose gels were used. PAGE (Fig. 1B) was chosen as the post-PCR electrophoresis protocol to assess genotype frequencies in individual L 3 (Table 1). For all samples, each band appeared double in PAGE (Fig. 1B), even in homozygous individuals and clones, because of the denaturation protocol employed, highlighting the importance of using positive controls for accurate genotyping through banding patterns after electrophoresis.
Using pooled L 3 from H. contortus isolates, PCR followed by 4% agarose gel electrophoresis showed amplification of a single fragment (177 bp) in the two susceptible isolates, whereas it resulted in banding patterns of various lengths (171 bp, 177 bp, and 180 bp) in resistant isolates. By sequencing cloned PCR products, a 6-bp deletion (leading to the 171 bp fragment) and a 3-bp insertion (leading to the 180 bp fragment) were detected. The two detected indels may potentially be responsible for the monepantel resistance status, as they were not detected in susceptible isolates, even in the SFD1 isolate that was resistant to other drugs than monepantel. Additionally, these indels result in amino acid changes in TMD2 of MPTL-1. In the nAChRs of free-living nematodes, TMD2 donates residues that line the ion channel (Jones and Sattelle 2003), thereby affecting protein function. Similarly, Bagnall et al. (2017) reported that most resistance-related mutations detected in mptl-1 led to TMD loss and truncated protein production, indicating that there may be a link between mutations in mptl-1, loss of MPTL-1 function, and monepantel resistance.
In addition, it is worth mentioning that the electrophoresis protocol on 4% agarose gels (modified from Bhattacharya and Van Meir 2019) following PCR detected fragments differing in only a few nucleotides (6 bp and 3 bp) in the pooled L 3 when using small-sized agarose gels. Thus, it may be useful for the initial screening of monepantel resistance status in H. contortus populations using a few samples of pooled larvae. However, owing to the curving of the edges of the gel, known as the "smile effect," that occurred in the outer  lanes of large agarose gels during electrophoresis, it was not a reliable post-PCR assay to determine genotypic frequencies at large-scale using larger-size gels. Furthermore, for correct genotype attribution and frequency determination, a PCR-based assay should be followed by a more sensitive and discriminatory electrophoresis protocol, such as PAGE, or, alternatively, high-resolution capillary electrophoresis. As the developed assay using PCR followed by PAGE is cost effective and able to quantify low allelic frequencies of mutations, it fulfills some of the requirements of an ideal molecular test to diagnose resistance (Kotze et al. 2020).
Based on the results presented here, we can confirm that several different mutations in mptl-1 in H. contortus may be associated with the phenotype of monepantel resistance in the same population (Bagnall et al. 2017) or in different populations, as reported for Teladorsagia circumcincta (Turnbull et al. 2019), indicating that monepantel resistance is likely a quantitative trait. However, exon 11 of mptl-1 was particularly responsive to the selective pressure imposed by monepantel, as two different indels in the same region were observed in H. contortus-resistant isolates from two different sources (field derived and in vivo-selected) and in an independent resistant field-derived population. In addition, the importance of the exon 11 region was previously reported in the literature. Kaminsky et al. (2008) described the absence of PCR amplification of intron 10 and exon 11 of mptl-1 in resistant individuals. Furthermore, downregulation of expression was detected in an AAD-mutant isolate by analyzing mRNA levels with primers directed to exons 11 and 12 of mptl-1 (Rufener et al. 2009). Thus, the developed assay was able to identify polymorphic patterns in exon 11 of mptl-1 potentially associated with the monepantel-resistant phenotype in H. contortus.

Conclusion
In conclusion, previous results obtained through genomic mapping for monepantel resistance were validated and a suitable PCR-based assay was designed for detecting indel polymorphisms in exon 11 of mptl-1 in H. contortus. Considering the use of Brazilian-derived isolates and the high genetic diversity of H. contortus observed worldwide, additional studies using H. contortus populations from other regions should be performed to verify the associations of the described indels in exon 11 of mptl-1 with the phenotype of monepantel resistance and thus validate them as molecular markers.

Supplementary Information
The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s00436-022-07682-6. Data availability Nucleotide sequence data reported in this paper are available in GenBank under accession numbers ON014540 to ON014573.

Declarations
Ethics approval This study was performed in strict accordance with the relevant guidelines and regulations of animal welfare in experimental science and with approval from the Animal Ethics Committee of Embrapa Pecuária Sudeste (Permit Number: 03/2019).

Conflict of interest
The authors declare no competing interests.