Transcriptional profiling and functional characterization of the Hc-NHR-49 gene in ivermectin resistance of Haemonchus contortus

Transcriptional profiling and functional characterization of the Hc-NHR-49 gene in ivermectin resistance of Haemonchus contortus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transcriptional profiling and functional characterization of the Hc-NHR-49 gene in ivermectin resistance of Haemonchus contortus Zeshuang Li, Penglong Wang, Xiaoping Luo, Gaowa Gong, Bin Li, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8472203/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background The barber’s pole worm (BPW), Haemonchus contortus , poses a significant threat to sheep health and livestock husbandry. Control has historically relied on synthetic anthelmintics such as ivermectin; however, widespread resistance to this drug has emerged. The functional role of nuclear hormone receptors (NHRs) in H. contortus remains poorly understood. Methods The Hc-NHR-49 gene was PCR-amplified and subjected to bioinformatic analysis. Expression profiles of the gene in resistant and sensitive strains across different developmental stages and under ivermectin stress were examined using RT‑qPCR. Polyclonal antibodies were generated in mice via recombinant prokaryotic expression and validated by Western blot. The spatial expression pattern of Hc‑NHR‑49 was further determined by immunohistochemistry. Finally, RNA interference followed by larval head swing assays was performed to assess its functional role in ivermectin response. Results In this study, we cloned and characterized the nuclear hormone receptor gene Hc-NHR-49 from H. contortus . The full-length cDNA is 1,272 bp, encoding a 423-amino-acid protein. Bioinformatics analysis revealed that Hc-NHR-49 is highly conserved across species, suggesting functional similarity. Recombinant Hc-NHR-49 was expressed and purified. Polyclonal antibodies raised in mice specifically recognized native Hc-NHR-49 in somatic extracts, as confirmed by Western blot. Immunohistochemical localization showed that Hc-NHR-49 is widely distributed, with particularly high expression in the intestine, uterus, ovaries and testes. Transcript levels were detected throughout all developmental stages in both ivermectin-susceptible and -resistant strains. To investigate its association with ivermectin resistance, worms were exposed to the EC₅₀ of ivermectin, which elicited a plastic expression response of Hc-NHR-49 . RNAi-mediated knockdown of Hc-NHR-49 increased ivermectin susceptibility in resistant parasites. Conclusions Collectively, our results suggest that Hc-NHR-49 is implicated in ivermectin resistance in H. contortus . These findings contribute to a deeper understanding of resistance mechanisms and could inform the future development of alternative control measures. Haemonchus contortus Nuclear hormone receptors Ivermectin Resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Haemonchus contortus , a hematophagous gastrointestinal nematode, parasitizes the abomasum of ruminants and is responsible for substantial global economic losses in livestock production [ 1 ]. The extensive and often indiscriminate use of anthelmintics has inevitably led to the widespread emergence of drug resistance worldwide, severely compromising the efficacy of chemical control against nematodes. Anthelmintic resistance (AR) to H. contortus has been extensively documented. For instance, a survey across 46 farms in eight southern U.S. states revealed that populations of H. contortus exhibited resistance to benzimidazole, levamisole, ivermectin (IVM) and moxidectin on 98%, 54%, 76% and 24% of the farms, respectively [ 2 ]. In northern Brazil, fecal egg count reduction tests were conducted on ten farms, which indicated that 37.1% of the pastures harbored nematode populations resistant to ivermectin [ 3 ]. Larval development assays demonstrated a high prevalence of resistance in gastrointestinal nematodes ( H. contortus and Trichostrongylus colubriformis ) from Colombian goats and sheep to albendazole, ivermectin, moxidectin and levamisole [ 4 ]. In eastern Inner Mongolia, China, a study of ten grazing sheep farms reported an average gastrointestinal nematode infection rate of 79.2% (range: 45–100%) and a mean fecal egg count of 1,813 eggs per gram (range: 0–32,400), with evidence of varying levels of resistance to avermectin, ivermectin and albendazole [ 5 ]. Despite the significant global threat posed by anthelmintic resistance in gastrointestinal nematodes, the precise molecular and genetic mechanisms driving this phenomenon remain incompletely understood. Recent research has established that enhanced enzymatic detoxification and target-site insensitivity are the principal mechanisms underlying anthelmintic resistance in nematodes [ 6 ]. Among the various documented pathways, elevated levels of anthelmintic resistance were frequently correlated with the activity of metabolic detoxification enzymes. P-glycoproteins ( P-gps ), belonging to the ATP-binding cassette (ABC) transporter family, function as efflux pumps that expel hydrophobic xenobiotics from cells, thereby reducing intracellular concentrations of anthelmintics [ 7 , 8 ]. For example, Caenorhabditis elegans with knocked-out P-gps showed increased sensitivity to IVM following anthelmintic exposure, confirming the involvement of these transporters in resistance [ 9 ]. In H. contortus , strains that survive under IVM pressure exhibit upregulation of P-gp-2 and P-gp-9 genes [ 10 ]. Similarly, previous studies have associated glutathione S-transferase (GST) with resistance. Inhibition of GST activity in H. contortus resulted in a significant reduction in larval motility and feeding, ultimately increasing susceptibility to ivermectin [ 11 ]. While numerous studies have linked detoxification enzymes to altered ivermectin sensitivity in nematodes, the specific transcription factors that regulate the expression of these genes remain largely uncharacterized. Nuclear hormone receptors (NHRs), members of the nuclear receptor superfamily, function as transcription factors that regulate signaling pathways and gene expression by binding lipophilic ligands [ 12 ]. They play a vital role in diverse biological processes, including xenobiotic metabolism, development and reproduction [ 13 ]. In H. contortus , a blood-feeding nematode, 40 NHR genes were found to respond to host serum in vitro, with one ( Hc-NHR-64 ) consistently activated by anthelmintic exposure [ 14 ]. A high-throughput RNA interference (RNAi) screen targeted 43 NHR genes in H. contortus and identified at least two essential genes: Hc-NHR-105 , crucial for viability, and Hc-NHR-17 , necessary for larval development in vitro [ 14 ]. Recent studies have indicated that Ce-NHR-8 in C. elegans regulates ivermectin resistance by upregulating IVM detoxification genes. Similarly, RNAi silencing of Hc-NHR-8 in larvae increased IVM sensitivity in both susceptible and resistant strains of H. contortus , suggesting functional conservation between Hc-NHR-8 and Ce-NHR-8 [ 15 ]. Furthermore, Hc-NHR-49 , a regulator of lipid metabolism and the oxidative stress response in nematodes, has also been linked to ivermectin resistance. Genome-wide association studies have identified Hc-NHR-49 as a candidate gene associated with IVM resistance in H. contortus [ 16 , 17 ]. However, this gene has not yet been fully characterized in the parasite, and the relationship between its expression dynamics and the progression of resistance remains unclear. Therefore, the identification of Hc-NHR-49 in H. contortus will elucidate of ivermectin resistance in this species and may inform the development of targeted control strategies. Using a previously established transcriptomic database for H. contortus , we identified the Hc-NHR-49 gene by querying the database with NHRs search terms. This study presents the cloning, recombinant expression and immunohistochemical staining of Hc-NHR-49 . Moreover, to investigate its role in anthelmintic resistance, we analyzed the transcriptional response of Hc-NHR-49 to ivermectin exposure and employed RNAi to knockdown its mRNA in an ivermectin-resistant strain. 2. Materials and menthods 2.1. Parasites The susceptible population of H. contortus (YCHc-022) was originally collected in 2018 from a farm in the Yuci District of Shanxi, China. The ivermectin-resistant population (CYHHc-136) was collected in the same year from a farm in the Ulanqab District of Inner Mongolia, China. These populations have been maintained in the laboratory for over six years without exposure to any anthelmintic. The ivermectin resistance status of these populations was confirmed through the fecal egg count reduction test, performed as previously described [ 18 ]. 2.2. Sample collection In preparation for H. contortus infection, rectal fecal samples were collected from sheep, and the McMaster technique was used to confirm that all sheep were free of nematode eggs. After a one-week adaptation period, sheep were divided into groups and orally inoculated with 5,000 infectious third-stage larvae (L3). One group received L3 from a susceptible strain, and the other received L3 from a resistant strain. Approximately 21 days post-infection, fecal samples from the infected sheep were collected and incubated at 27℃ for 7 days to allow larval development to L3. The L3 larvae were then collected using a Baermann apparatus [ 19 ], thoroughly washed with deionized water, and stored at 16℃ until use. Egg collection Fresh fecal samples were homogenized on the day of collection and mixed with saturated saline solution. The mixture was sequentially filtered through sieves with mesh sizes of 20, 40, 100, 200 and 300. Nematode eggs were then recovered using the saturated saline flotation method. Subsequently, the collected liquid was filtered through a 500-mesh sieve, followed by rinsing the sieve with physiological saline to collect the wash solution into a beaker. After allowing the wash solution to settle for 20 min, the supernatant was discarded, and the egg suspension was transferred to a 15 mL centrifuge tube. The suspension was left to stand at room temperature for 15 min, after which the supernatant was discarded. This washing step was repeated until the eggs were thoroughly cleaned. Larval culture Distilled water (1–2 mL) was added to each well of a 6-well plate, followed by an appropriate amount of nematode eggs. After incubation at 27°C for 36 h, first-stage larvae (L1) were collected. The remaining cultures were further incubated at 27°C for 3 days, after which second-stage larvae (L2) were collected, and stored at -80°C until use. Adult collection According to the requirements of the Animal Welfare Regulations of the Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences, sheep infected with a susceptible strain (YCHc-022) and a resistant strain (CYHHc-136) were dissected. The abomasum was collected, and fresh adult parasites were carefully extracted using a fine needle. Following rinsing with distilled water, the collected parasites were promptly preserved in liquid nitrogen or 4% paraformaldehyde for subsequent use. 2.3. Total RNA extraction and reverse transcription Total RNA was extracted from 10 adult female nematodes of the YCHc-022 strain for amplification of full-length Hc-NHR-49 cDNA. Briefly, nematodes were homogenized in TRIzol Reagent (15596026, invitrogen, US) using an OSE-Y20 tissue homogenizer (Tiangen Biotech, China), following the manufacturer's protocols. The extracted RNA was dissolved in 30 µL of DEPC-treated water, quantified by measuring the absorbance at 260 nm, and assessed for purity using the A260/A280 ratio. Reverse transcription was performed with the TransScript One-step gDNA Remover and cDNA Synthesis SuperMix (R323, Vazyme Biotech, China). The resulting cDNA was stored at -20°C for subsequent use. 2.4. Cloning of Hc-NHR-49 gene and sequence analysis The full-length Hc-NHR-49 cDNA was amplified by PCR using gene-specific primers (Table S1 ), which were designed from our in-house H. contortus transcriptome database. The PCR conditions were as follows: initial denaturation at 94°C for 5 min; 35 cycles of 94°C for 15 s, 50°C for 15 s and 68°C for 30 s. The PCR products were purified with the TIANgel Midi Purification Kit (TIANGEN, China) and cloned into the pEASY®-Blunt Cloning Vector (TransGen, China) according to the manufacturer's protocols. Conserved protein domains were identified by multiple amino acid sequence alignment using Clustal Omega. For phylogenetic analysis, the nucleotide sequence of Hc-NHR-49 was aligned with homologous sequences from other species retrieved from GenBank, and a phylogenetic tree was constructed in MEGA 11 using the neighbor-joining method with 1000 bootstrap replicates. The Hc-NHR-49 sequence has been deposited in GenBank under accession number PX701906. 2.5. Transcriptional analysis of Hc-NHR-49 in H. contortus 2.5.1. Analysis of Hc-NHR-49 expression in different development stages To examine the stage- and strain-specific expression of Hc-NHR-49 , samples were collected from both ivermectin-susceptible (YCHc-022) and -resistant strains (CYHHc-136) of H. contortus . The developmental stages analyzed included adult females, adult males, larvae and eggs. For each biological replicate, the sample consisted of 10 adults (male or female), 5000 larvae, or 10000 eggs per strain. This sampling was performed in triplicate. Total RNA was then extracted from each sample (20 µL) and reverse transcribed (1 µg RNA) for cDNA synthesis. The transcriptional levels of Hc-NHR-49 across different stages were quantified by RT-qPCR, as described in Section 2.6 . 2.5.2. Ivermectin-induced expression of the Hc-NHR-49 gene To evaluate the expression of Hc-NHR-49 in response to ivermectin in both resistant and susceptible strains, approximately 5,000 L3 larvae per strain were collected. They were then exposed separately to either ivermectin at their respective EC₅₀ concentrations or to 0.5% DMSO (vehicle control). Larvae were harvested at 12, 24, 36 and 48 h post-exposure and transferred to 1.5 mL centrifuge tubes. Each treatment was performed in triplicate. Finally, the transcript levels of Hc-NHR-49 in the collected samples were quantified by RT-qPCR as described in Section 2.6 . 2.6. Real-time quantitative PCR The expression of Hc-NHR-49 across different life stages, strains and after ivermectin induction was quantified by RT-qPCR using a CFX96™ Real-Time PCR System (Applied Biosystems). Gene-specific primers were designed with Primer 5.0 (Table S1 ). Each 20 µL reaction contained 10 µL of TransStart Tip Green qPCR Super Mix, 7.2 µL of ddH₂O, 0.4 µL each of forward and reverse primers (10 µM), and 2 µL of cDNA template. The thermal cycling protocol consisted of an initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Fluorescence was acquired at the end of the 60°C extension step. A melt curve analysis (60°C to 95°C) was performed to confirm amplicon specificity. Three independent biological replicates were analyzed for each sample, with GAPDH used as the endogenous control gene [ 20 ]. Relative gene expression was calculated using the 2 −ΔΔCT method [ 21 ]. 2.7. Expression and purification of the recombinant Hc-NHR-49 The Hc-NHR-49 gene was synthesized and cloned into the pCold-TF expression vector via XhoI and Hindlll restriction sites (Table S1 ). The resulting recombinant plasmid, pCold-TF-Hc-NHR-49, was transformed into Escherichia coli BL21 (DE3) competent cells. Protein expression was induced by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubating at 16°C for 24 h. Cells were then harvested, lysed and centrifuged. The soluble recombinant Hc-NHR-49 (rHc-NHR-49) protein was purified from the supernatant using Ni-NTA Agarose (AS045, ABclonal, China) according to the manufacturer's protocols. The purified protein was analyzed by SDS-PAGE, quantified with a BCA protein assay kit (PC0020, Solarbio, China), aliquoted and stored at -80°C for subsequent use. 2.8. Polyclonal antibodies and western blot analysis The reactivity and specificity of the mouse antiserum were analyzed by western blot. Samples containing 50 ng of purified rHc-NHR-49 or 10 µg of soluble nematode extract were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% (w/v) skimmed milk in PBST (PBS with 0.1% Tween-20) for 1 h at room temperature. They were then incubated with the primary mouse antiserum at a 1:500 dilution in blocking buffer for 1 h. After washing, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (HS201,TransGen, China) at a 1:10000 dilution for 1 h. Following additional washes, immunoreactive bands were detected using an enhanced chemiluminescence (ECL) substrate (180–5001, Tanon, China) according to the manufacturer's instructions. 2.9. Immunohistochemical localization of Hc-NHR-49 in H. contortus tissues Fresh adult female and male nematodes were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed by heating the sections in citrate buffer (pH 6.0). Subsequently, endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide at room temperature for 25 min in the dark, followed by three 5 min washes in PBS (pH 7.4). Non-specific binding was blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature. After blocking, sections were incubated overnight at 4°C with the primary antibody diluted 1:800 in PBS within a humidified chamber. Following three 5-min washes in PBS, sections were incubated for 50 min at room temperature with HRP-conjugated goat anti-mouse secondary antibody (1:5000). After another three washes in PBS, immunoreactivity was visualized by applying freshly prepared 3,3′-diaminobenzidine (DAB) chromogen to the sections. The DAB reaction was monitored under a light microscope and stopped when optimal signal-to-noise contrast was achieved, with positive staining appearing as a brown-yellow precipitate. Sections were then counterstained, dehydrated, cleared and mounted for microscopic examination. 2.10. RNAi in nematodes Larval preparation and exsheathment Active third-stage larvae of the ivermectin-resistant (CYHHc-136) strains were used. Approximately 5,000 L3 were exsheathed by incubation in 0.6% sodium hypochlorite (NaClO) at 36°C for 50 min with periodic gentle inversion. Complete exsheathment was confirmed by microscopic examination. The exsheathed larvae were then washed three times with 0.9% NaCl solution, followed by three rinses in diethylpyrocarbonate (DEPC)-treated water. Production of dsRNA in E. coli Double-stranded RNAs (dsRNAs) targeting the gene of interest (dsHc-NHR-49) and green fluorescent protein (GFP; control) were expressed using the L4440 vector system. The L4440 plasmid contains two convergent T7 promoters flanking a multiple cloning site and an ampicillin resistance marker [ 22 ]. Target fragments were amplified by PCR with gene-specific primers (Table S1 ) and engineered with XhoI and NotI sites for directional cloning into L4440. The resulting recombinant plasmids (L4440-dsHc-NHR-49 and L4440-dsGFP) were verified by sequencing. Expression and extraction The verified plasmids were independently transformed into the RNase III-deficient E. coli HT115 (DE3) strain, which is incapable of degrading dsRNA. Positive transformants were selected on LB agar plates containing ampicillin. A single colony for each construct was inoculated into LB broth and cultured at 37°C with shaking (200 rpm). When the optical density at 600 nm (OD₆₀₀) reached 0.5, dsRNA expression was induced by adding IPTG to a final concentration of 1 mM, followed by incubation for an additional 4 h. Bacterial cells were harvested by centrifugation, and dsRNA was extracted from the cell pellets using TRIzol® Reagent according to the manufacturer's instructions. The GFP sequence used for the control dsRNA corresponds to GenBank accession MN623123.1. RNAi by dsRNA soaking RNAi was performed using a dsRNA soaking method. For each treatment group (dsHc-NHR-49 or dsGFP control), three independent biological replicates were set up, with each replicate consisting of 5,000 resistant larvae. The L3 were transferred to sterile 1.5 mL tubes and incubated with 100 µL of nuclease-free water containing 2000 ng/µL of the respective dsRNA. The tubes were incubated at 4°C for 12 h to allow for dsRNA uptake. After incubation, the dsRNA solution was removed. Total RNA was then extracted from randomly selected larvae per replicate, reverse-transcribed into cDNA, and subjected to RT-qPCR to evaluate Hc-NHR-49 knockdown efficiency. 2.11. Motility assay Following RNAi, the treated L3 larvae were collected, washed, and resuspended in sterile water. Approximately 20 to 50 larvae (in a 10 µL aliquot) were transferred to each well of a new 24-well cell culture plate. To each well, 1,700 µL of sterile water and 90 µL of ivermectin stock solution were added to achieve a final concentration of 13.97 nmol/L. Control wells received an equivalent volume of 0.5% DMSO. The plates were incubated at 37°C for 24 h. After incubation, larval movement was assessed by counting the number of head swings over a 1 min observation period for ten randomly selected larvae per well. The entire experiment was performed with three independent biological replicates. 2.12. Statistical analysis All statistical analyses were performed using GraphPad Prism software (version 8.0.0 for Windows, GraphPad Software, USA). Data are presented as the mean ± standard error of the mean (SEM) from at least three independent biological replicates. Differences between two groups were analyzed using the non-parametric Mann-Whitney U test. A p value of less than 0.05 ( p < 0.05) was considered statistically significant. 3. Results 3.1. Amplification, cloning and sequencing of Hc-NHR-49 The coding sequence of Hc-NHR-49 comprised 1,272 bp, encoding a protein of 423 amino acids with a theoretical molecular weight of 47 kDa and an isoelectric point (pI) of 7.87. No putative signal peptides or transmembrane domains were predicted by SignalP-5.0 and TMHMM-2.0 servers. A putative nuclear localization signal (NLS) motif, KRSR (residues 94–97), was identified within the sequence “GSSPTKRSRGS” (starting at position 91). A DNA-binding domain (residues 13–88) and a ligand-binding domain (residues 158–351) were identified (Fig. 1 ). The C4-type steroid receptor zinc finger signature motif (residues 13–33) and steroid hormone receptor signatures (residues 74–84, 172–193, 193–209, 266–281 and 326–343) were highly conserved. Phylogenetic analysis demonstrated that Hc-NHR-49 shared high sequence identity with orthologs from Toxocara canis (85%), Caenorhabditis elegans (82.4%) and Caenorhabditis japonica (83%) (Fig. 2 ). 3.2. Transcriptional levels of Hc-NHR-49 at different life stages in susceptible and resistant strains The transcript of Hc-NHR-49 was detected at all life stages (including eggs, L1, L2, L3, adult males and females) in both susceptible and resistant strains of H. contortus using RT-qPCR. In the susceptible strains, the expression level of Hc-NHR-49 was not significantly different in the L2 stage compared with eggs (Fig. 3 A). However, it was significantly lower in other stages, with expression levels reduced to approximately 41% ( p = 0.0003), 65% ( p = 0.0001), 49% ( p = 0.0001) and 66% ( p = 0.0001) of those in eggs for the L1, L3, female and male stages, respectively (Fig. 3 A). In contrast, in the resistant strains, Hc-NHR-49 expression was significantly upregulated (5.44-fold, p = 0.0001) only in the L2 stage relative to eggs (Fig. 3 B). Conversely, it was significantly downregulated in the L3 and male stages, with expression levels 58% ( p = 0.0002) and 80% ( p = 0.0001) of those in eggs, respectively. No significant differences were observed in the remaining stages (L1 and female). 3.3. Inductive effects of Hc-NHR-49 under the treatment of ivermectin To further expore the relationship between the Hc-NHR-49 and ivermectin resistance in H. contortus , ivermectin at EC 50 was used to induce their expression in both susceptible and resistant strains. The expression levels of Hc-NHR-49 after ivermectin exposure were detected at different times. In the susceptible strain (Fig. 4 A), Hc-NHR-49 expression showed a biphasic response. It was significantly downregulated at 12 h and 48 h (to 74% and 36% of the control level, respectively; both p = 0.007), but exhibited a transient 2.36-fold upregulation at 24 h ( p = 0.009) before returning to baseline by 36 h. In contrast, the resistant strain (Fig. 4 B) displayed a simpler pattern: no change at 12 h, followed by a significant 1.68-fold upregulation at 24 h ( p = 0.006), and a return to control levels by 36 h. 3.4. Recombinant Hc-NHR-49 expression, purification and polyclonal antibody detection The recombinant Hc-NHR-49 protein was expressed in E. coli BL21 (DE3) cells induced by IPTG. SDS-PAGE analysis of bacterial lysates revealed a predominant induced band at ~ 47 kDa, with the majority of the protein present in the soluble supernatant fraction (Fig. 5 ). This soluble fraction was subsequently subjected to Ni-NTA affinity chromatography, yielding a single purified band at the expected size (Fig. 5 ). To evaluate antibody specificity, Western blot analysis was performed using mouse anti-rHc-NHR-49 antiserum against both the purified recombinant protein and soluble somatic extracts from H. contortus . As shown in Fig. 6 , the antiserum specifically recognized a single band corresponding to the recombinant protein and a native protein of comparable size in the nematode extracts. The observed molecular weight of rHc-NHR-49 (~ 96 kDa) exceeds its calculated weight based on the amino acid sequence, which is attributable to the 49 kDa fusion tag from the pCold-TF vector. 3.5. Localization of Hc-NHR-49 in H. contortus To visualize the distribution of Hc-NHR-49, immunohistochemical staining was conducted on sections of both female and male H. contortus . Consecutive 5 µm sections were incubated with mouse anti-Hc-NHR-49 serum. This revealed specific Hc-NHR-49 expression in female reproductive organs - the uterus (ut) and ovaries (ov) - and the intestine (int) (Fig. 7 A). In males, expression was localized to the testes (te), cement gland (gl.cem) and intestine (Fig. 7 B). Notably, intestinal staining intensity was markedly higher in both sexes, suggesting abundant Hc-NHR-49 protein in this tissue. 3.6. Knockdown of Hc-NHR-49 increases ivermectin toxicity To investigate the functional role of Hc-NHR-49 in ivermectin resistance, we knocked down its expression via RNAi in the resistant strain and assessed ivermectin toxicity. Treatment with 2000 ng/µL of dsHc-NHR-49 significantly reduced the expression of the target gene to 57% of the level in the dsGFP control group ( p = 0.0001; Fig. 8 ). Consequently, when exposed to ivermectin at the EC 50 concentration for the resistant strain, the head thrash frequency of the dsHc-NHR-49-treated worms was significantly reduced to 21.97/min, compared to 59.37/min in the dsGFP control group ( p = 0.001; Fig. 9 ). 4. Discussion Nuclear hormone receptors are ligand-modulated transcription factors that respond to steroids, fatty acid-like molecules and other compounds[ 23 ]. Beyond its established roles, the NHR-49 has emerged as a critical regulator of stress responses, directly modulating specific genes and pathways. However, the molecular and biochemical properties of NHR-49 in the parasitic nematode H. contortus remain largely uncharacterized. In this study, we identified and characterized NHR-49 in H. contortus for the first time. The cDNA was cloned, sequenced, and subjected to bioinformatic alignment with publicly available sequences from GenBank. Compared with NHR-49 from Homo sapiens , Mus musculus , Rattus norvegicus , Xenopus tropicalis and C. elegans , these domains are highly conserved. Phylogenetic analysis based on amino acid sequences showed that Hc-NHR-49 is most closely related to that of C. elegans (82.4% sequence identity), while sequence identities with Toxocara canis and Caenorhabditis japonica were 85% and 83%, respectively, further confirming that NHR-49 is highly conserved across a variety of species. These results also suggest that Hc-NHR-49 has similar functions to HNR-49 of these species. The functions of NHR-49 are likely tissue-specific and may vary among species. In the model nematode C. elegans , somatic NHR-49 enables adaptation to physiological stress by up regulating genes involved in mitochondrial β-oxidation and fatty-acid desaturation [ 24 ]. In the parasitic nematode H. contortus , we found that Hc-NHR-49 has a distinct and widespread tissue distribution, with strong expression in metabolically and reproductively active tissues—including the intestine, uterus, ovaries, testes, and cement gland. This ubiquitous expression pattern, contrasting with the more specific somatic role in C. elegans , indicates that Hc-NHR-49 may fulfill multiple biological functions in the parasite, potentially extending beyond metabolic regulation. In the model nematode C. elegans , NHR-49 regulates mitochondrial/peroxisomal β-oxidation and fatty acid desaturation processes critical for development and adaptation to nutrient stress [ 25 , 26 ]. NHR-49 loss-of-function results in metabolic dysregulation, reduced lifespan and impaired survival under starvation [ 27 ]. Furthermore, it is required in specific germline stem cells for reproductive recovery after prolonged fasting [ 28 ]. Collectively, NHR-49 emerges as a master regulator that promotes survival across diverse physiological contexts by reprogramming metabolism and restoring lipid homeostasis. Given this pivotal role in stress adaptation, we hypothesized that its ortholog in parasitic nematodes, Hc-NHR-49 , might be involved in anthelmintic resistance. To test this, we analyzed Hc-NHR-49 expression in H. contortus . We show that Hc-NHR-49 expression varies throughout development in both ivermectin-susceptible and -resistant strains and, crucially, is dynamically regulated following ivermectin exposure. These findings extend the conserved role of Hc-NHR-49 in stress responses to parasitic nematodes and specifically implicate Hc-NHR-49 in the complex process of ivermectin resistance acquisition. Although a previous genome-wide association study (GWAS) based on whole-genome resequencing identified Hc-NHR-49 as a candidate gene for ivermectin resistance in H. contortus [ 17 ], its functional role remained unverified. Here, we employed RNAi to silence Hc-NHR-49 in H. contortus . Following Hc-NHR-49 knockdown and IVM treatment, a significant reduction in larval motility (head thrash frequency) was observed compared to controls. This finding indicates that Hc-NHR-49 silencing enhances parasite sensitivity to IVM, thereby functionally validating its role as a key regulator of IVM susceptibility. This result aligns with the established functions of other nematode nuclear hormone receptors in anthelmintic response. For example, loss of NHR-8 in C. elegans increases sensitivity to IVM [ 15 ], and NHR-176 regulates the metabolism of anthelmintics in this model organism [ 29 ]. Furthermore, the same GWAS [ 17 ] implicated several other Hc-NHR genes (e.g., Hc-NHR-3 , Hc-NHR -14 and Hc-NHR -22) in IVM detoxification. In contrast, IVM susceptibility is unaffected by the loss of NHR-48 or DAF-12 in C. elegans [ 30 ], highlighting the specificity of NHR functions. Collectively, our functional data establishes Hc-NHR-49 as a pivotal regulator of ivermectin resistance in H. contortus , where its targeted functional impairment re-sensitizes resistant parasites to the drug. The overexpression of xenobiotic detoxification genes is a pleiotropic mechanism contributing to anthelmintic resistance in nematodes [ 31 ]. This includes the upregulation of ABC efflux transporters, which enhance anthelmintic elimination upon exposure to compounds like ivermectin, thereby conferring resistance [ 32 ]. Consistently, detoxification metabolic enzymes are also overexpressed in IVM-resistant nematodes [ 8 , 33 , 34 ], with specific genes such as GST-4 and Pgp-9 implicated in ivermectin resistance in both H. contortus and C. elegans [ 35 , 36 ]. The transcriptional regulation of these detoxification pathways is often mediated by nuclear hormone receptors. In C. elegans , for instance, NHR-176 regulates cyp-35d1 , a gene controlling the metabolism of thiabendazole [ 29 ]. Similarly, and of direct relevance to ivermectin resistance, deletion of NHR-8 in nematodes significantly reduces the expression of key detoxification genes (including Pgps , CYPs and GSTs ) and increases susceptibility to ivermectin, demonstrating that NHR-8 is a master transcriptional regulator of detoxification and a determinant of anthelmintic sensitivity [ 37 ]. This regulatory paradigm mirrors the “xenosensor” function of nuclear receptors in mammals, which orchestrate detoxification gene expression in response to xenobiotics. Given the established role of NHRs in governing detoxification and anthelmintic resistance, and based on our expression and functional data, we propose that Hc-NHR-49 confers ivermectin resistance in H. contortus by regulating the expression of detoxification enzyme genes. 5. Conclusion In summary, we report the first functional characterization of Hc-NHR-49 in H. contortus , establishing its role in ivermectin resistance. We characterized the gene and produced a specific polyclonal antibody to determine its tissue-specific expression. Transcriptional profiling revealed dynamic expression of Hc-NHR-49 both during development and in response to ivermectin exposure. Most importantly, functional analysis using RNAi-mediated knockdown and a phenotypic motility assay demonstrated that Hc-NHR-49 contributes to ivermectin resistance, likely by regulating detoxification pathways. Abbreviations NHRs nuclear hormone receptors BPW barber’s pole worm AR anthelmintic resistance IVM ivermectin P-gps P-glycoproteins ABC ATP-binding cassette GST glutathione S-transferase RNAi RNA interference YCHc-022 susceptible population of H. contortus CYHHc-136 ivermectin-resistant population of H. contortus L3 third-stage larvae L2 second-stage larvae L1 first-stage larvae EC 50 half maximal effective concentration RT-qPCR quantitative reverse transcription polymerase chain reaction IPTG isopropyl β-D-1-thiogalactopyranoside SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis PVDF polyvinylidene difluoride PBST phosphate-Buffered Saline with Tween® 20 DAB 3,3′-diaminobenzidine dsRNAs Double-stranded RNAs GFP green fluorescent protein DMSO Dimethyl Sulfoxide NLS nuclear localization signal GWAS genome-wide association study DAF Decay-accelerating factor CYPs Cytochromes P450. Declarations Ethics approval and consent to participate This study was approved by the Animal Ethics Committee of the Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences (2024-0010). All sheep were handled in strict accordance with good animal practices according to the Animal Ethics Procedures and Guidelines of the People’s Republic of China. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. Funding This work was supported by the National Natural Science Foundation of China (no. 32160844 and 32260892), Inner Mongolia Natural Science Foundation Project (no. 2025YQ021) and Xinjiang Uygur Autonomous Region Tianshan Innovation Team (no. 2023TSYCTD0008). Author Contribution ZeShuang Li and Penglong Wang participated in data interpretation and manuscript drafting. Xiaoping Luo and Gawa Gong jointly conceptualized the study, contributed to the study design and data interpretation, and assisted in manuscript preparation. Bin Li conducted statistical analyses and critically revised the manuscript. Yaning Li, Luyang Tang and Xuesen Zhang contributed to the collection of various life stages of *Haemonchus contortus* , including eggs, first-stage larvae, second-stage larvae, third-stage larvae and adults. Dandan Liu conducted statistical analyses and critically revised the manuscript. Jiuru Huangfu provided language editing and manuscript proof reading. Wei Zhang and Junyan Li contributed to the study design and participated in critical revisions of the manuscript. All authors have read and approved the final version of the manuscript. Acknowledgement We would like to thank all the persons who provided their kind help and suggestions for this work and the manuscript. 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08:12:05","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15946,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8472203/v1/2d67ccff655a7f757d52b801.png"},{"id":100372846,"identity":"282edd5d-b8a4-499f-943f-a3f4a73ceacc","added_by":"auto","created_at":"2026-01-16 08:13:16","extension":"xml","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":135884,"visible":true,"origin":"","legend":"","description":"","filename":"d8c6d0b156b147b7afc218ebcffbe5fd1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8472203/v1/5a7c780060cfd5ac1b888f1c.xml"},{"id":100265341,"identity":"ed6c8a1d-9c4f-4337-b411-ba6fa4052353","added_by":"auto","created_at":"2026-01-14 18:09:20","extension":"html","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":158638,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8472203/v1/98ab22f00bab863abbeba9e3.html"},{"id":100265303,"identity":"bc67353a-675b-495d-8e2f-5baccfe20a43","added_by":"auto","created_at":"2026-01-14 18:09:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":762706,"visible":true,"origin":"","legend":"\u003cp\u003eAlignment of the amino acid sequences of the NHR-49\u003cem\u003e \u003c/em\u003efrom \u003cem\u003eToxocara canis\u003c/em\u003e (A0AOB2UP53), \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e (E5QCI8), \u003cem\u003eHomo sapiens \u003c/em\u003e(P41235), \u003cem\u003eMus musculus\u003c/em\u003e (Q9WUU6), \u003cem\u003eDanio rerio\u003c/em\u003e (Q8AXB6), \u003cem\u003eDiploscapter pachys\u003c/em\u003e(A0A2A2K1B9), \u003cem\u003eParascaris univalens\u003c/em\u003e (A0A915BRC7) and \u003cem\u003eAscaris suum\u003c/em\u003e(F1KZG6). The C4-type steroid receptor zinc finger signature motif is highlighted in blue. The steroid hormone receptor signatures is highlighted in green. The nuclear localization signal is highlighted in pink.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8472203/v1/1ec3941f5a0b27d0c1fd7e11.jpg"},{"id":100372904,"identity":"a7816ec7-5960-4e63-8744-11461e34946f","added_by":"auto","created_at":"2026-01-16 08:13:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33470,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbour-joining analysis of the \u003cem\u003eNHR-49\u003c/em\u003e from \u003cem\u003eToxocara canis\u003c/em\u003e (A0AOB2UP53), \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e(E5QCI8), \u003cem\u003eHomo sapiens \u003c/em\u003e(P41235), \u003cem\u003eMus musculus\u003c/em\u003e (Q9WUU6), \u003cem\u003eDanio rerio\u003c/em\u003e (Q8AXB6), \u003cem\u003eDiploscapter pachys\u003c/em\u003e (A0A2A2K1B9), \u003cem\u003eParascaris univalens\u003c/em\u003e (A0A915BRC7), \u003cem\u003eAscaris suum\u003c/em\u003e (F1KZG6), \u003cem\u003eStrongylus vulgaris\u003c/em\u003e (A0A3P7IHR1), \u003cem\u003eNippostrongylus brasiliensis \u003c/em\u003e(A0A158R0T7), \u003cem\u003eXenopus tropicalis\u003c/em\u003e (A0A6I8QP06), \u003cem\u003eCaenorhabditis japonica \u003c/em\u003e(A0A8R1E0I5) and \u003cem\u003eDrosophila melanogaster\u003c/em\u003e (P49866). Evolutionary distances were computed using the Poisson correction method. Branch support values (1000 bootstraps) for nodes are indicated.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8472203/v1/e33daccc632923ef205b8b21.jpg"},{"id":100372541,"identity":"8ffa21f5-b2ab-4130-b077-c91252d30d65","added_by":"auto","created_at":"2026-01-16 08:12:39","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":138014,"visible":true,"origin":"","legend":"\u003cp\u003eRT-qPCR expression analysis of the \u003cem\u003eHc-NHR-49\u003c/em\u003e gene in different life stages in susceptible and resistant strains. (A) Susceptible strains (YCHc-022). (B) Resistant strains (CYHHc-136). Error bars represent the standard error of the calculated mean based on three biological replicates. Different letters on the error bars show significant differences of the \u003cem\u003eHc-NHR-49\u003c/em\u003e gene among different developmental stages (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8472203/v1/4b930a6266629aa307fd1818.jpg"},{"id":100372608,"identity":"04611633-78fa-41e4-b1e8-5bce59b28c30","added_by":"auto","created_at":"2026-01-16 08:12:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":114643,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression levels of \u003cem\u003eHc-NHR-49\u003c/em\u003e gene in L3 under the treatment of ivermectin. (A) EC\u003csub\u003e50\u003c/sub\u003e treatment in susceptible strains (YCHc-022); (B) EC\u003csub\u003e50\u003c/sub\u003e treatment in resistant strains (CYHHc-136). Error bars represent the standard error of the calculated mean based on three biological replicates. Different asterisks on the error bars show significant differences of the \u003cem\u003eHc-NHR-49\u003c/em\u003e gene between induction and control (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8472203/v1/134cf489122351a428da9132.jpg"},{"id":100372545,"identity":"840b4787-4c35-486a-8ed2-be5db94d277e","added_by":"auto","created_at":"2026-01-16 08:12:39","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":34968,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE analysis of recombinant Hc-NHR-49 from \u003cem\u003eH. contortus\u003c/em\u003e. M: The molecular size marker, 1: purified recombinant Hc-NHR-49 protein, 2: \u003cem\u003eE.coli\u003c/em\u003e lysate control (harboring only the pCold-TF expression vector). 3: Bacterial lysate of recombinant protein before induction. 4: bacterial lysate supernatant of recombinant protein after induction by IPTG. 5: bacterial lysate precipitation of recombinant protein after induction.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8472203/v1/6482ba7287471c831db9c530.jpg"},{"id":100372169,"identity":"7367a23a-ab5d-43c5-a030-4d9db2ae885e","added_by":"auto","created_at":"2026-01-16 08:11:48","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":54563,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot analysis of purified rHc-NHR-49 and soluble nematode extraction. Anti-rHc-NHR-49 serum recognized purified rHc-NHR-49 and native Hc-NHR-49. M: prestained protein ladder. 1: purified rHc-NHR-49 proteins were identified with mouse polyclonal anti-rHc-NHR-49 serum (1:500). 2: natural Hc-NHR-49 proteins of \u003cem\u003eH. contortus\u003c/em\u003e were identified with mouse polyclonal anti-rHc-NHR-49 serum (1:500).\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8472203/v1/81ba98fb7daa78a154391907.jpg"},{"id":100371184,"identity":"ba0a2006-6c23-4395-a454-e12abb9f073c","added_by":"auto","created_at":"2026-01-16 08:09:35","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":90710,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical localization of Hc-NHR-49 in female and male \u003cem\u003eH. contortus\u003c/em\u003e. A: Female, the uterus (ut), ovaries (ov) and intestine (int). B: Male, the testes (te), cement gland (gl.cem) and intestine.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8472203/v1/3dd247ca0c07f02138575d43.jpg"},{"id":100372494,"identity":"2b45425e-eaf2-4f12-bcd7-290af3218652","added_by":"auto","created_at":"2026-01-16 08:12:30","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":28980,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of \u003cem\u003eHc-NHR-49\u003c/em\u003e genedetermined by RT-qPCR in the resistant strains of L3 after RNAi. Error bars represent the standard error of the calculated means based on three biological replicates. Different asterisks on the error bars show significant differences of \u003cem\u003eHc-NHR-49\u003c/em\u003e gene between dsGFP and dsHc-NHR-49 groups (*** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8472203/v1/c5dd6c9a2c8827fbc6ff79aa.jpg"},{"id":100372427,"identity":"ce8e85db-3c98-4ea0-b0d0-43a82e60a95c","added_by":"auto","created_at":"2026-01-16 08:12:21","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":24051,"visible":true,"origin":"","legend":"\u003cp\u003eHead thrash frequence of the resistant \u003cem\u003eH. contortus\u003c/em\u003e after silence of \u003cem\u003eHc-NHR-49\u003c/em\u003e and exploration to ivermectin at EC\u003csub\u003e50\u003c/sub\u003e. Error bars represent the standard errors of the calculated means based on three biological replicates. Different asterisks on the error bars show significant differences of head thrash frequence between dsGFP and dsHc-NHR-49 groups (*** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Picture9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8472203/v1/e02b91d1e9a199dfc8a61071.jpg"},{"id":100421950,"identity":"a48fcd57-6ec6-4f68-9455-711b72713117","added_by":"auto","created_at":"2026-01-16 14:03:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2510449,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8472203/v1/c05d1be4-1c95-41a0-b2ca-94eb3479118c.pdf"},{"id":100265307,"identity":"57175c95-50cb-42a3-ba20-3b2854d7fcbb","added_by":"auto","created_at":"2026-01-14 18:09:20","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":13161,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8472203/v1/beffc08e6d40b189c4cff5d3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transcriptional profiling and functional characterization of the Hc-NHR-49 gene in ivermectin resistance of Haemonchus contortus","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eHaemonchus contortus\u003c/em\u003e, a hematophagous gastrointestinal nematode, parasitizes the abomasum of ruminants and is responsible for substantial global economic losses in livestock production [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The extensive and often indiscriminate use of anthelmintics has inevitably led to the widespread emergence of drug resistance worldwide, severely compromising the efficacy of chemical control against nematodes. Anthelmintic resistance (AR) to \u003cem\u003eH. contortus\u003c/em\u003e has been extensively documented. For instance, a survey across 46 farms in eight southern U.S. states revealed that populations of \u003cem\u003eH. contortus\u003c/em\u003e exhibited resistance to benzimidazole, levamisole, ivermectin (IVM) and moxidectin on 98%, 54%, 76% and 24% of the farms, respectively [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In northern Brazil, fecal egg count reduction tests were conducted on ten farms, which indicated that 37.1% of the pastures harbored nematode populations resistant to ivermectin [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Larval development assays demonstrated a high prevalence of resistance in gastrointestinal nematodes (\u003cem\u003eH. contortus\u003c/em\u003e and \u003cem\u003eTrichostrongylus colubriformis\u003c/em\u003e) from Colombian goats and sheep to albendazole, ivermectin, moxidectin and levamisole [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In eastern Inner Mongolia, China, a study of ten grazing sheep farms reported an average gastrointestinal nematode infection rate of 79.2% (range: 45\u0026ndash;100%) and a mean fecal egg count of 1,813 eggs per gram (range: 0\u0026ndash;32,400), with evidence of varying levels of resistance to avermectin, ivermectin and albendazole [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Despite the significant global threat posed by anthelmintic resistance in gastrointestinal nematodes, the precise molecular and genetic mechanisms driving this phenomenon remain incompletely understood.\u003c/p\u003e \u003cp\u003eRecent research has established that enhanced enzymatic detoxification and target-site insensitivity are the principal mechanisms underlying anthelmintic resistance in nematodes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Among the various documented pathways, elevated levels of anthelmintic resistance were frequently correlated with the activity of metabolic detoxification enzymes. P-glycoproteins (\u003cem\u003eP-gps\u003c/em\u003e), belonging to the ATP-binding cassette (ABC) transporter family, function as efflux pumps that expel hydrophobic xenobiotics from cells, thereby reducing intracellular concentrations of anthelmintics [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. For example, \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e with knocked-out \u003cem\u003eP-gps\u003c/em\u003e showed increased sensitivity to IVM following anthelmintic exposure, confirming the involvement of these transporters in resistance [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In \u003cem\u003eH. contortus\u003c/em\u003e, strains that survive under IVM pressure exhibit upregulation of \u003cem\u003eP-gp-2\u003c/em\u003e and \u003cem\u003eP-gp-9\u003c/em\u003e genes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Similarly, previous studies have associated glutathione S-transferase (GST) with resistance. Inhibition of GST activity in \u003cem\u003eH. contortus\u003c/em\u003e resulted in a significant reduction in larval motility and feeding, ultimately increasing susceptibility to ivermectin [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. While numerous studies have linked detoxification enzymes to altered ivermectin sensitivity in nematodes, the specific transcription factors that regulate the expression of these genes remain largely uncharacterized.\u003c/p\u003e \u003cp\u003eNuclear hormone receptors (NHRs), members of the nuclear receptor superfamily, function as transcription factors that regulate signaling pathways and gene expression by binding lipophilic ligands [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. They play a vital role in diverse biological processes, including xenobiotic metabolism, development and reproduction [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In \u003cem\u003eH. contortus\u003c/em\u003e, a blood-feeding nematode, 40 NHR genes were found to respond to host serum in vitro, with one (\u003cem\u003eHc-NHR-64\u003c/em\u003e) consistently activated by anthelmintic exposure [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. A high-throughput RNA interference (RNAi) screen targeted 43 NHR genes in \u003cem\u003eH. contortus\u003c/em\u003e and identified at least two essential genes: \u003cem\u003eHc-NHR-105\u003c/em\u003e, crucial for viability, and \u003cem\u003eHc-NHR-17\u003c/em\u003e, necessary for larval development in vitro [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Recent studies have indicated that \u003cem\u003eCe-NHR-8\u003c/em\u003e in \u003cem\u003eC. elegans\u003c/em\u003e regulates ivermectin resistance by upregulating IVM detoxification genes. Similarly, RNAi silencing of \u003cem\u003eHc-NHR-8\u003c/em\u003e in larvae increased IVM sensitivity in both susceptible and resistant strains of \u003cem\u003eH. contortus\u003c/em\u003e, suggesting functional conservation between \u003cem\u003eHc-NHR-8\u003c/em\u003e and \u003cem\u003eCe-NHR-8\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, \u003cem\u003eHc-NHR-49\u003c/em\u003e, a regulator of lipid metabolism and the oxidative stress response in nematodes, has also been linked to ivermectin resistance. Genome-wide association studies have identified \u003cem\u003eHc-NHR-49\u003c/em\u003e as a candidate gene associated with IVM resistance in \u003cem\u003eH. contortus\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, this gene has not yet been fully characterized in the parasite, and the relationship between its expression dynamics and the progression of resistance remains unclear.\u003c/p\u003e \u003cp\u003eTherefore, the identification of \u003cem\u003eHc-NHR-49\u003c/em\u003e in \u003cem\u003eH. contortus\u003c/em\u003e will elucidate of ivermectin resistance in this species and may inform the development of targeted control strategies. Using a previously established transcriptomic database for \u003cem\u003eH. contortus\u003c/em\u003e, we identified the \u003cem\u003eHc-NHR-49\u003c/em\u003e gene by querying the database with NHRs search terms. This study presents the cloning, recombinant expression and immunohistochemical staining of \u003cem\u003eHc-NHR-49\u003c/em\u003e. Moreover, to investigate its role in anthelmintic resistance, we analyzed the transcriptional response of \u003cem\u003eHc-NHR-49\u003c/em\u003e to ivermectin exposure and employed RNAi to knockdown its mRNA in an ivermectin-resistant strain.\u003c/p\u003e"},{"header":"2. Materials and menthods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Parasites\u003c/h2\u003e \u003cp\u003eThe susceptible population of \u003cem\u003eH. contortus\u003c/em\u003e (YCHc-022) was originally collected in 2018 from a farm in the Yuci District of Shanxi, China. The ivermectin-resistant population (CYHHc-136) was collected in the same year from a farm in the Ulanqab District of Inner Mongolia, China. These populations have been maintained in the laboratory for over six years without exposure to any anthelmintic. The ivermectin resistance status of these populations was confirmed through the fecal egg count reduction test, performed as previously described [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Sample collection\u003c/h2\u003e \u003cp\u003eIn preparation for \u003cem\u003eH. contortus\u003c/em\u003e infection, rectal fecal samples were collected from sheep, and the McMaster technique was used to confirm that all sheep were free of nematode eggs. After a one-week adaptation period, sheep were divided into groups and orally inoculated with 5,000 infectious third-stage larvae (L3). One group received L3 from a susceptible strain, and the other received L3 from a resistant strain. Approximately 21 days post-infection, fecal samples from the infected sheep were collected and incubated at 27℃ for 7 days to allow larval development to L3. The L3 larvae were then collected using a Baermann apparatus [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], thoroughly washed with deionized water, and stored at 16℃ until use.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEgg collection\u003c/strong\u003e \u003cp\u003eFresh fecal samples were homogenized on the day of collection and mixed with saturated saline solution. The mixture was sequentially filtered through sieves with mesh sizes of 20, 40, 100, 200 and 300. Nematode eggs were then recovered using the saturated saline flotation method. Subsequently, the collected liquid was filtered through a 500-mesh sieve, followed by rinsing the sieve with physiological saline to collect the wash solution into a beaker. After allowing the wash solution to settle for 20 min, the supernatant was discarded, and the egg suspension was transferred to a 15 mL centrifuge tube. The suspension was left to stand at room temperature for 15 min, after which the supernatant was discarded. This washing step was repeated until the eggs were thoroughly cleaned.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eLarval culture\u003c/strong\u003e \u003cp\u003eDistilled water (1\u0026ndash;2 mL) was added to each well of a 6-well plate, followed by an appropriate amount of nematode eggs. After incubation at 27\u0026deg;C for 36 h, first-stage larvae (L1) were collected. The remaining cultures were further incubated at 27\u0026deg;C for 3 days, after which second-stage larvae (L2) were collected, and stored at -80\u0026deg;C until use.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAdult collection\u003c/strong\u003e \u003cp\u003e According to the requirements of the Animal Welfare Regulations of the Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences, sheep infected with a susceptible strain (YCHc-022) and a resistant strain (CYHHc-136) were dissected. The abomasum was collected, and fresh adult parasites were carefully extracted using a fine needle. Following rinsing with distilled water, the collected parasites were promptly preserved in liquid nitrogen or 4% paraformaldehyde for subsequent use.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Total RNA extraction and reverse transcription\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from 10 adult female nematodes of the YCHc-022 strain for amplification of full-length \u003cem\u003eHc-NHR-49\u003c/em\u003e cDNA. Briefly, nematodes were homogenized in TRIzol Reagent (15596026, invitrogen, US) using an OSE-Y20 tissue homogenizer (Tiangen Biotech, China), following the manufacturer's protocols. The extracted RNA was dissolved in 30 \u0026micro;L of DEPC-treated water, quantified by measuring the absorbance at 260 nm, and assessed for purity using the A260/A280 ratio. Reverse transcription was performed with the TransScript One-step gDNA Remover and cDNA Synthesis SuperMix (R323, Vazyme Biotech, China). The resulting cDNA was stored at -20\u0026deg;C for subsequent use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cloning of \u003cem\u003eHc-NHR-49\u003c/em\u003e gene and sequence analysis\u003c/h2\u003e \u003cp\u003eThe full-length \u003cem\u003eHc-NHR-49\u003c/em\u003e cDNA was amplified by PCR using gene-specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), which were designed from our in-house \u003cem\u003eH. contortus\u003c/em\u003e transcriptome database. The PCR conditions were as follows: initial denaturation at 94\u0026deg;C for 5 min; 35 cycles of 94\u0026deg;C for 15 s, 50\u0026deg;C for 15 s and 68\u0026deg;C for 30 s. The PCR products were purified with the TIANgel Midi Purification Kit (TIANGEN, China) and cloned into the pEASY\u0026reg;-Blunt Cloning Vector (TransGen, China) according to the manufacturer's protocols. Conserved protein domains were identified by multiple amino acid sequence alignment using Clustal Omega. For phylogenetic analysis, the nucleotide sequence of \u003cem\u003eHc-NHR-49\u003c/em\u003e was aligned with homologous sequences from other species retrieved from GenBank, and a phylogenetic tree was constructed in MEGA 11 using the neighbor-joining method with 1000 bootstrap replicates. The \u003cem\u003eHc-NHR-49\u003c/em\u003e sequence has been deposited in GenBank under accession number PX701906.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Transcriptional analysis of \u003cem\u003eHc-NHR-49\u003c/em\u003e in \u003cem\u003eH. contortus\u003c/em\u003e\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Analysis of \u003cem\u003eHc-NHR-49\u003c/em\u003e expression in different development stages\u003c/h2\u003e \u003cp\u003eTo examine the stage- and strain-specific expression of \u003cem\u003eHc-NHR-49\u003c/em\u003e, samples were collected from both ivermectin-susceptible (YCHc-022) and -resistant strains (CYHHc-136) of \u003cem\u003eH. contortus\u003c/em\u003e. The developmental stages analyzed included adult females, adult males, larvae and eggs. For each biological replicate, the sample consisted of 10 adults (male or female), 5000 larvae, or 10000 eggs per strain. This sampling was performed in triplicate. Total RNA was then extracted from each sample (20 \u0026micro;L) and reverse transcribed (1 \u0026micro;g RNA) for cDNA synthesis. The transcriptional levels of \u003cem\u003eHc-NHR-49\u003c/em\u003e across different stages were quantified by RT-qPCR, as described in Section \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003e2.6\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. Ivermectin-induced expression of the \u003cem\u003eHc-NHR-49\u003c/em\u003e gene\u003c/h2\u003e \u003cp\u003eTo evaluate the expression of \u003cem\u003eHc-NHR-49\u003c/em\u003e in response to ivermectin in both resistant and susceptible strains, approximately 5,000 L3 larvae per strain were collected. They were then exposed separately to either ivermectin at their respective EC₅₀ concentrations or to 0.5% DMSO (vehicle control). Larvae were harvested at 12, 24, 36 and 48 h post-exposure and transferred to 1.5 mL centrifuge tubes. Each treatment was performed in triplicate. Finally, the transcript levels of \u003cem\u003eHc-NHR-49\u003c/em\u003e in the collected samples were quantified by RT-qPCR as described in Section \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003e2.6\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Real-time quantitative PCR\u003c/h2\u003e \u003cp\u003eThe expression of \u003cem\u003eHc-NHR-49\u003c/em\u003e across different life stages, strains and after ivermectin induction was quantified by RT-qPCR using a CFX96\u0026trade; Real-Time PCR System (Applied Biosystems). Gene-specific primers were designed with Primer 5.0 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Each 20 \u0026micro;L reaction contained 10 \u0026micro;L of TransStart Tip Green qPCR Super Mix, 7.2 \u0026micro;L of ddH₂O, 0.4 \u0026micro;L each of forward and reverse primers (10 \u0026micro;M), and 2 \u0026micro;L of cDNA template. The thermal cycling protocol consisted of an initial denaturation at 95\u0026deg;C for 30 s, followed by 40 cycles of 95\u0026deg;C for 10 s and 60\u0026deg;C for 30 s. Fluorescence was acquired at the end of the 60\u0026deg;C extension step. A melt curve analysis (60\u0026deg;C to 95\u0026deg;C) was performed to confirm amplicon specificity. Three independent biological replicates were analyzed for each sample, with \u003cem\u003eGAPDH\u003c/em\u003e used as the endogenous control gene [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Relative gene expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Expression and purification of the recombinant Hc-NHR-49\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eHc-NHR-49\u003c/em\u003e gene was synthesized and cloned into the pCold-TF expression vector via \u003cem\u003eXhoI\u003c/em\u003e and \u003cem\u003eHindlll\u003c/em\u003e restriction sites (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The resulting recombinant plasmid, pCold-TF-Hc-NHR-49, was transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 (DE3) competent cells. Protein expression was induced by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubating at 16\u0026deg;C for 24 h. Cells were then harvested, lysed and centrifuged. The soluble recombinant Hc-NHR-49 (rHc-NHR-49) protein was purified from the supernatant using Ni-NTA Agarose (AS045, ABclonal, China) according to the manufacturer's protocols. The purified protein was analyzed by SDS-PAGE, quantified with a BCA protein assay kit (PC0020, Solarbio, China), aliquoted and stored at -80\u0026deg;C for subsequent use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Polyclonal antibodies and western blot analysis\u003c/h2\u003e \u003cp\u003eThe reactivity and specificity of the mouse antiserum were analyzed by western blot. Samples containing 50 ng of purified rHc-NHR-49 or 10 \u0026micro;g of soluble nematode extract were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% (w/v) skimmed milk in PBST (PBS with 0.1% Tween-20) for 1 h at room temperature. They were then incubated with the primary mouse antiserum at a 1:500 dilution in blocking buffer for 1 h. After washing, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (HS201,TransGen, China) at a 1:10000 dilution for 1 h. Following additional washes, immunoreactive bands were detected using an enhanced chemiluminescence (ECL) substrate (180\u0026ndash;5001, Tanon, China) according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Immunohistochemical localization of Hc-NHR-49 in \u003cem\u003eH. contortus\u003c/em\u003e tissues\u003c/h2\u003e \u003cp\u003eFresh adult female and male nematodes were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed by heating the sections in citrate buffer (pH 6.0). Subsequently, endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide at room temperature for 25 min in the dark, followed by three 5 min washes in PBS (pH 7.4). Non-specific binding was blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature. After blocking, sections were incubated overnight at 4\u0026deg;C with the primary antibody diluted 1:800 in PBS within a humidified chamber. Following three 5-min washes in PBS, sections were incubated for 50 min at room temperature with HRP-conjugated goat anti-mouse secondary antibody (1:5000). After another three washes in PBS, immunoreactivity was visualized by applying freshly prepared 3,3\u0026prime;-diaminobenzidine (DAB) chromogen to the sections. The DAB reaction was monitored under a light microscope and stopped when optimal signal-to-noise contrast was achieved, with positive staining appearing as a brown-yellow precipitate. Sections were then counterstained, dehydrated, cleared and mounted for microscopic examination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.10. RNAi in nematodes\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eLarval preparation and exsheathment\u003c/strong\u003e \u003cp\u003eActive third-stage larvae of the ivermectin-resistant (CYHHc-136) strains were used. Approximately 5,000 L3 were exsheathed by incubation in 0.6% sodium hypochlorite (NaClO) at 36\u0026deg;C for 50 min with periodic gentle inversion. Complete exsheathment was confirmed by microscopic examination. The exsheathed larvae were then washed three times with 0.9% NaCl solution, followed by three rinses in diethylpyrocarbonate (DEPC)-treated water.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eProduction of dsRNA in \u003cem\u003eE. coli\u003c/em\u003e\u003c/strong\u003e \u003cp\u003eDouble-stranded RNAs (dsRNAs) targeting the gene of interest (dsHc-NHR-49) and green fluorescent protein (GFP; control) were expressed using the L4440 vector system. The L4440 plasmid contains two convergent T7 promoters flanking a multiple cloning site and an ampicillin resistance marker [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Target fragments were amplified by PCR with gene-specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and engineered with \u003cem\u003eXhoI\u003c/em\u003e and \u003cem\u003eNotI\u003c/em\u003e sites for directional cloning into L4440. The resulting recombinant plasmids (L4440-dsHc-NHR-49 and L4440-dsGFP) were verified by sequencing.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eExpression and extraction\u003c/strong\u003e \u003cp\u003eThe verified plasmids were independently transformed into the RNase III-deficient \u003cem\u003eE. coli\u003c/em\u003e HT115 (DE3) strain, which is incapable of degrading dsRNA. Positive transformants were selected on LB agar plates containing ampicillin. A single colony for each construct was inoculated into LB broth and cultured at 37\u0026deg;C with shaking (200 rpm). When the optical density at 600 nm (OD₆₀₀) reached 0.5, dsRNA expression was induced by adding IPTG to a final concentration of 1 mM, followed by incubation for an additional 4 h. Bacterial cells were harvested by centrifugation, and dsRNA was extracted from the cell pellets using TRIzol\u0026reg; Reagent according to the manufacturer's instructions. The GFP sequence used for the control dsRNA corresponds to GenBank accession MN623123.1.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eRNAi by dsRNA soaking\u003c/strong\u003e \u003cp\u003eRNAi was performed using a dsRNA soaking method. For each treatment group (dsHc-NHR-49 or dsGFP control), three independent biological replicates were set up, with each replicate consisting of 5,000 resistant larvae. The L3 were transferred to sterile 1.5 mL tubes and incubated with 100 \u0026micro;L of nuclease-free water containing 2000 ng/\u0026micro;L of the respective dsRNA. The tubes were incubated at 4\u0026deg;C for 12 h to allow for dsRNA uptake. After incubation, the dsRNA solution was removed. Total RNA was then extracted from randomly selected larvae per replicate, reverse-transcribed into cDNA, and subjected to RT-qPCR to evaluate \u003cem\u003eHc-NHR-49\u003c/em\u003e knockdown efficiency.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Motility assay\u003c/h2\u003e \u003cp\u003eFollowing RNAi, the treated L3 larvae were collected, washed, and resuspended in sterile water. Approximately 20 to 50 larvae (in a 10 \u0026micro;L aliquot) were transferred to each well of a new 24-well cell culture plate. To each well, 1,700 \u0026micro;L of sterile water and 90 \u0026micro;L of ivermectin stock solution were added to achieve a final concentration of 13.97 nmol/L. Control wells received an equivalent volume of 0.5% DMSO. The plates were incubated at 37\u0026deg;C for 24 h. After incubation, larval movement was assessed by counting the number of head swings over a 1 min observation period for ten randomly selected larvae per well. The entire experiment was performed with three independent biological replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using GraphPad Prism software (version 8.0.0 for Windows, GraphPad Software, USA). Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) from at least three independent biological replicates. Differences between two groups were analyzed using the non-parametric Mann-Whitney U test. A \u003cem\u003ep\u003c/em\u003e value of less than 0.05 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Amplification, cloning and sequencing of \u003cem\u003eHc-NHR-49\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe coding sequence of \u003cem\u003eHc-NHR-49\u003c/em\u003e comprised 1,272 bp, encoding a protein of 423 amino acids with a theoretical molecular weight of 47 kDa and an isoelectric point (pI) of 7.87. No putative signal peptides or transmembrane domains were predicted by SignalP-5.0 and TMHMM-2.0 servers. A putative nuclear localization signal (NLS) motif, KRSR (residues 94\u0026ndash;97), was identified within the sequence \u0026ldquo;GSSPTKRSRGS\u0026rdquo; (starting at position 91). A DNA-binding domain (residues 13\u0026ndash;88) and a ligand-binding domain (residues 158\u0026ndash;351) were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The C4-type steroid receptor zinc finger signature motif (residues 13\u0026ndash;33) and steroid hormone receptor signatures (residues 74\u0026ndash;84, 172\u0026ndash;193, 193\u0026ndash;209, 266\u0026ndash;281 and 326\u0026ndash;343) were highly conserved. Phylogenetic analysis demonstrated that Hc-NHR-49 shared high sequence identity with orthologs from \u003cem\u003eToxocara canis\u003c/em\u003e (85%), \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e (82.4%) and \u003cem\u003eCaenorhabditis japonica\u003c/em\u003e (83%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Transcriptional levels of \u003cem\u003eHc-NHR-49\u003c/em\u003e at different life stages in susceptible and resistant strains\u003c/h2\u003e \u003cp\u003eThe transcript of \u003cem\u003eHc-NHR-49\u003c/em\u003e was detected at all life stages (including eggs, L1, L2, L3, adult males and females) in both susceptible and resistant strains of \u003cem\u003eH. contortus\u003c/em\u003e using RT-qPCR. In the susceptible strains, the expression level of \u003cem\u003eHc-NHR-49\u003c/em\u003e was not significantly different in the L2 stage compared with eggs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, it was significantly lower in other stages, with expression levels reduced to approximately 41% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0003), 65% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001), 49% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001) and 66% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001) of those in eggs for the L1, L3, female and male stages, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In contrast, in the resistant strains, \u003cem\u003eHc-NHR-49\u003c/em\u003e expression was significantly upregulated (5.44-fold, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001) only in the L2 stage relative to eggs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Conversely, it was significantly downregulated in the L3 and male stages, with expression levels 58% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002) and 80% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001) of those in eggs, respectively. No significant differences were observed in the remaining stages (L1 and female).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Inductive effects of \u003cem\u003eHc-NHR-49\u003c/em\u003e under the treatment of ivermectin\u003c/h2\u003e \u003cp\u003eTo further expore the relationship between the \u003cem\u003eHc-NHR-49\u003c/em\u003e and ivermectin resistance in \u003cem\u003eH. contortus\u003c/em\u003e, ivermectin at EC\u003csub\u003e50\u003c/sub\u003e was used to induce their expression in both susceptible and resistant strains. The expression levels of \u003cem\u003eHc-NHR-49\u003c/em\u003e after ivermectin exposure were detected at different times. In the susceptible strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), \u003cem\u003eHc-NHR-49\u003c/em\u003e expression showed a biphasic response. It was significantly downregulated at 12 h and 48 h (to 74% and 36% of the control level, respectively; both \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007), but exhibited a transient 2.36-fold upregulation at 24 h (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009) before returning to baseline by 36 h. In contrast, the resistant strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) displayed a simpler pattern: no change at 12 h, followed by a significant 1.68-fold upregulation at 24 h (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006), and a return to control levels by 36 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Recombinant \u003cem\u003eHc-NHR-49\u003c/em\u003e expression, purification and polyclonal antibody detection\u003c/h2\u003e \u003cp\u003eThe recombinant Hc-NHR-49 protein was expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) cells induced by IPTG. SDS-PAGE analysis of bacterial lysates revealed a predominant induced band at ~\u0026thinsp;47 kDa, with the majority of the protein present in the soluble supernatant fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This soluble fraction was subsequently subjected to Ni-NTA affinity chromatography, yielding a single purified band at the expected size (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). To evaluate antibody specificity, Western blot analysis was performed using mouse anti-rHc-NHR-49 antiserum against both the purified recombinant protein and soluble somatic extracts from \u003cem\u003eH. contortus\u003c/em\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the antiserum specifically recognized a single band corresponding to the recombinant protein and a native protein of comparable size in the nematode extracts. The observed molecular weight of rHc-NHR-49 (~\u0026thinsp;96 kDa) exceeds its calculated weight based on the amino acid sequence, which is attributable to the 49 kDa fusion tag from the pCold-TF vector.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Localization of Hc-NHR-49 in \u003cem\u003eH. contortus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo visualize the distribution of Hc-NHR-49, immunohistochemical staining was conducted on sections of both female and male \u003cem\u003eH. contortus\u003c/em\u003e. Consecutive 5 \u0026micro;m sections were incubated with mouse anti-Hc-NHR-49 serum. This revealed specific Hc-NHR-49 expression in female reproductive organs - the uterus (ut) and ovaries (ov) - and the intestine (int) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). In males, expression was localized to the testes (te), cement gland (gl.cem) and intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Notably, intestinal staining intensity was markedly higher in both sexes, suggesting abundant Hc-NHR-49 protein in this tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Knockdown of \u003cem\u003eHc-NHR-49\u003c/em\u003e increases ivermectin toxicity\u003c/h2\u003e \u003cp\u003eTo investigate the functional role of \u003cem\u003eHc-NHR-49\u003c/em\u003e in ivermectin resistance, we knocked down its expression via RNAi in the resistant strain and assessed ivermectin toxicity. Treatment with 2000 ng/\u0026micro;L of dsHc-NHR-49 significantly reduced the expression of the target gene to 57% of the level in the dsGFP control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Consequently, when exposed to ivermectin at the EC\u003csub\u003e50\u003c/sub\u003e concentration for the resistant strain, the head thrash frequency of the dsHc-NHR-49-treated worms was significantly reduced to 21.97/min, compared to 59.37/min in the dsGFP control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eNuclear hormone receptors are ligand-modulated transcription factors that respond to steroids, fatty acid-like molecules and other compounds[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Beyond its established roles, the \u003cem\u003eNHR-49\u003c/em\u003e has emerged as a critical regulator of stress responses, directly modulating specific genes and pathways. However, the molecular and biochemical properties of \u003cem\u003eNHR-49\u003c/em\u003e in the parasitic nematode \u003cem\u003eH. contortus\u003c/em\u003e remain largely uncharacterized. In this study, we identified and characterized \u003cem\u003eNHR-49\u003c/em\u003e in \u003cem\u003eH. contortus\u003c/em\u003e for the first time. The cDNA was cloned, sequenced, and subjected to bioinformatic alignment with publicly available sequences from GenBank. Compared with \u003cem\u003eNHR-49\u003c/em\u003e from \u003cem\u003eHomo sapiens\u003c/em\u003e, \u003cem\u003eMus musculus\u003c/em\u003e, \u003cem\u003eRattus norvegicus\u003c/em\u003e, \u003cem\u003eXenopus tropicalis\u003c/em\u003e and \u003cem\u003eC. elegans\u003c/em\u003e, these domains are highly conserved. Phylogenetic analysis based on amino acid sequences showed that Hc-NHR-49 is most closely related to that of \u003cem\u003eC. elegans\u003c/em\u003e (82.4% sequence identity), while sequence identities with \u003cem\u003eToxocara canis\u003c/em\u003e and \u003cem\u003eCaenorhabditis japonica\u003c/em\u003e were 85% and 83%, respectively, further confirming that NHR-49 is highly conserved across a variety of species. These results also suggest that Hc-NHR-49 has similar functions to HNR-49 of these species.\u003c/p\u003e \u003cp\u003eThe functions of NHR-49 are likely tissue-specific and may vary among species. In the model nematode \u003cem\u003eC. elegans\u003c/em\u003e, somatic NHR-49 enables adaptation to physiological stress by up regulating genes involved in mitochondrial β-oxidation and fatty-acid desaturation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In the parasitic nematode \u003cem\u003eH. contortus\u003c/em\u003e, we found that Hc-NHR-49 has a distinct and widespread tissue distribution, with strong expression in metabolically and reproductively active tissues\u0026mdash;including the intestine, uterus, ovaries, testes, and cement gland. This ubiquitous expression pattern, contrasting with the more specific somatic role in \u003cem\u003eC. elegans\u003c/em\u003e, indicates that Hc-NHR-49 may fulfill multiple biological functions in the parasite, potentially extending beyond metabolic regulation.\u003c/p\u003e \u003cp\u003eIn the model nematode \u003cem\u003eC. elegans\u003c/em\u003e, \u003cem\u003eNHR-49\u003c/em\u003e regulates mitochondrial/peroxisomal β-oxidation and fatty acid desaturation processes critical for development and adaptation to nutrient stress [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. \u003cem\u003eNHR-49\u003c/em\u003e loss-of-function results in metabolic dysregulation, reduced lifespan and impaired survival under starvation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Furthermore, it is required in specific germline stem cells for reproductive recovery after prolonged fasting [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Collectively, \u003cem\u003eNHR-49\u003c/em\u003e emerges as a master regulator that promotes survival across diverse physiological contexts by reprogramming metabolism and restoring lipid homeostasis. Given this pivotal role in stress adaptation, we hypothesized that its ortholog in parasitic nematodes, \u003cem\u003eHc-NHR-49\u003c/em\u003e, might be involved in anthelmintic resistance. To test this, we analyzed \u003cem\u003eHc-NHR-49\u003c/em\u003e expression in \u003cem\u003eH. contortus\u003c/em\u003e. We show that \u003cem\u003eHc-NHR-49\u003c/em\u003e expression varies throughout development in both ivermectin-susceptible and -resistant strains and, crucially, is dynamically regulated following ivermectin exposure. These findings extend the conserved role of \u003cem\u003eHc-NHR-49\u003c/em\u003e in stress responses to parasitic nematodes and specifically implicate \u003cem\u003eHc-NHR-49\u003c/em\u003e in the complex process of ivermectin resistance acquisition.\u003c/p\u003e \u003cp\u003eAlthough a previous genome-wide association study (GWAS) based on whole-genome resequencing identified \u003cem\u003eHc-NHR-49\u003c/em\u003e as a candidate gene for ivermectin resistance in \u003cem\u003eH. contortus\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], its functional role remained unverified. Here, we employed RNAi to silence \u003cem\u003eHc-NHR-49\u003c/em\u003e in \u003cem\u003eH. contortus\u003c/em\u003e. Following \u003cem\u003eHc-NHR-49\u003c/em\u003e knockdown and IVM treatment, a significant reduction in larval motility (head thrash frequency) was observed compared to controls. This finding indicates that \u003cem\u003eHc-NHR-49\u003c/em\u003e silencing enhances parasite sensitivity to IVM, thereby functionally validating its role as a key regulator of IVM susceptibility. This result aligns with the established functions of other nematode nuclear hormone receptors in anthelmintic response. For example, loss of \u003cem\u003eNHR-8\u003c/em\u003e in \u003cem\u003eC. elegans\u003c/em\u003e increases sensitivity to IVM [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and \u003cem\u003eNHR-176\u003c/em\u003e regulates the metabolism of anthelmintics in this model organism [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Furthermore, the same GWAS [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] implicated several other \u003cem\u003eHc-NHR\u003c/em\u003e genes (e.g., \u003cem\u003eHc-NHR-3\u003c/em\u003e, \u003cem\u003eHc-NHR\u003c/em\u003e-14 and \u003cem\u003eHc-NHR\u003c/em\u003e-22) in IVM detoxification. In contrast, IVM susceptibility is unaffected by the loss of \u003cem\u003eNHR-48\u003c/em\u003e or \u003cem\u003eDAF-12\u003c/em\u003e in \u003cem\u003eC. elegans\u003c/em\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], highlighting the specificity of NHR functions. Collectively, our functional data establishes \u003cem\u003eHc-NHR-49\u003c/em\u003e as a pivotal regulator of ivermectin resistance in \u003cem\u003eH. contortus\u003c/em\u003e, where its targeted functional impairment re-sensitizes resistant parasites to the drug.\u003c/p\u003e \u003cp\u003eThe overexpression of xenobiotic detoxification genes is a pleiotropic mechanism contributing to anthelmintic resistance in nematodes [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This includes the upregulation of ABC efflux transporters, which enhance anthelmintic elimination upon exposure to compounds like ivermectin, thereby conferring resistance [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Consistently, detoxification metabolic enzymes are also overexpressed in IVM-resistant nematodes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], with specific genes such as \u003cem\u003eGST-4\u003c/em\u003e and \u003cem\u003ePgp-9\u003c/em\u003e implicated in ivermectin resistance in both \u003cem\u003eH. contortus\u003c/em\u003e and \u003cem\u003eC. elegans\u003c/em\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The transcriptional regulation of these detoxification pathways is often mediated by nuclear hormone receptors. In \u003cem\u003eC. elegans\u003c/em\u003e, for instance, \u003cem\u003eNHR-176\u003c/em\u003e regulates \u003cem\u003ecyp-35d1\u003c/em\u003e, a gene controlling the metabolism of thiabendazole [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, and of direct relevance to ivermectin resistance, deletion of \u003cem\u003eNHR-8\u003c/em\u003e in nematodes significantly reduces the expression of key detoxification genes (including \u003cem\u003ePgps\u003c/em\u003e, \u003cem\u003eCYPs\u003c/em\u003e and \u003cem\u003eGSTs\u003c/em\u003e) and increases susceptibility to ivermectin, demonstrating that NHR-8 is a master transcriptional regulator of detoxification and a determinant of anthelmintic sensitivity [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This regulatory paradigm mirrors the \u0026ldquo;xenosensor\u0026rdquo; function of nuclear receptors in mammals, which orchestrate detoxification gene expression in response to xenobiotics. Given the established role of NHRs in governing detoxification and anthelmintic resistance, and based on our expression and functional data, we propose that \u003cem\u003eHc-NHR-49\u003c/em\u003e confers ivermectin resistance in \u003cem\u003eH. contortus\u003c/em\u003e by regulating the expression of detoxification enzyme genes.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, we report the first functional characterization of \u003cem\u003eHc-NHR-49\u003c/em\u003e in \u003cem\u003eH. contortus\u003c/em\u003e, establishing its role in ivermectin resistance. We characterized the gene and produced a specific polyclonal antibody to determine its tissue-specific expression. Transcriptional profiling revealed dynamic expression of \u003cem\u003eHc-NHR-49\u003c/em\u003e both during development and in response to ivermectin exposure. Most importantly, functional analysis using RNAi-mediated knockdown and a phenotypic motility assay demonstrated that \u003cem\u003eHc-NHR-49\u003c/em\u003e contributes to ivermectin resistance, likely by regulating detoxification pathways.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNHRs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enuclear hormone receptors\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBPW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebarber\u0026rsquo;s pole worm\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eanthelmintic resistance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIVM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eivermectin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP-gps\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eP-glycoproteins\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eABC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eATP-binding cassette\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglutathione S-transferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRNAi\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRNA interference\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eYCHc-022\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esusceptible population of \u003cem\u003eH. contortus\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCYHHc-136\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eivermectin-resistant population of \u003cem\u003eH. contortus\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eL3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethird-stage larvae\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eL2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esecond-stage larvae\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eL1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efirst-stage larvae\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEC\u003csub\u003e50\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehalf maximal effective concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT-qPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003equantitative reverse transcription polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIPTG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eisopropyl β-D-1-thiogalactopyranoside\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDS-PAGE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePVDF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolyvinylidene difluoride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate-Buffered Saline with Tween\u0026reg; 20\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3,3\u0026prime;-diaminobenzidine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003edsRNAs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDouble-stranded RNAs\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGFP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egreen fluorescent protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDimethyl Sulfoxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNLS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enuclear localization signal\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGWAS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egenome-wide association study\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDecay-accelerating factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCYPs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCytochromes P450.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eThis study was approved by the Animal Ethics Committee of the Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences (2024-0010). All sheep were handled in strict accordance with good animal practices according to the Animal Ethics Procedures and Guidelines of the People\u0026rsquo;s Republic of China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (no. 32160844 and 32260892), Inner Mongolia Natural Science Foundation Project (no. 2025YQ021) and Xinjiang Uygur Autonomous Region Tianshan Innovation Team (no. 2023TSYCTD0008).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eZeShuang Li and Penglong Wang participated in data interpretation and manuscript drafting. Xiaoping Luo and Gawa Gong jointly conceptualized the study, contributed to the study design and data interpretation, and assisted in manuscript preparation. Bin Li conducted statistical analyses and critically revised the manuscript. Yaning Li, Luyang Tang and Xuesen Zhang contributed to the collection of various life stages of *Haemonchus contortus* , including eggs, first-stage larvae, second-stage larvae, third-stage larvae and adults. Dandan Liu conducted statistical analyses and critically revised the manuscript. Jiuru Huangfu provided language editing and manuscript proof reading. Wei Zhang and Junyan Li contributed to the study design and participated in critical revisions of the manuscript. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe would like to thank all the persons who provided their kind help and suggestions for this work and the manuscript.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEmery DL, Hunt PW, Le Jambre LF. \u003cem\u003eHaemonchus contortus\u003c/em\u003e: the then and now, and where to from here? 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Antimicrob Agents Chemother. 2016;60 8:4809\u0026ndash;19; doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/aac.00713-16\u003c/span\u003e\u003cspan address=\"10.1128/aac.00713-16\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Haemonchus contortus, Nuclear hormone receptors, Ivermectin, Resistance","lastPublishedDoi":"10.21203/rs.3.rs-8472203/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8472203/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe barber\u0026rsquo;s pole worm (BPW), \u003cem\u003eHaemonchus contortus\u003c/em\u003e, poses a significant threat to sheep health and livestock husbandry. Control has historically relied on synthetic anthelmintics such as ivermectin; however, widespread resistance to this drug has emerged. The functional role of nuclear hormone receptors (NHRs) in \u003cem\u003eH. contortus\u003c/em\u003e remains poorly understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eHc-NHR-49\u003c/em\u003e gene was PCR-amplified and subjected to bioinformatic analysis. Expression profiles of the gene in resistant and sensitive strains across different developmental stages and under ivermectin stress were examined using RT‑qPCR. Polyclonal antibodies were generated in mice via recombinant prokaryotic expression and validated by Western blot. The spatial expression pattern of Hc‑NHR‑49 was further determined by immunohistochemistry. Finally, RNA interference followed by larval head swing assays was performed to assess its functional role in ivermectin response.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we cloned and characterized the nuclear hormone receptor gene Hc-NHR-49 from \u003cem\u003eH. contortus\u003c/em\u003e. The full-length cDNA is 1,272 bp, encoding a 423-amino-acid protein. Bioinformatics analysis revealed that Hc-NHR-49 is highly conserved across species, suggesting functional similarity. Recombinant Hc-NHR-49 was expressed and purified. Polyclonal antibodies raised in mice specifically recognized native Hc-NHR-49 in somatic extracts, as confirmed by Western blot. Immunohistochemical localization showed that Hc-NHR-49 is widely distributed, with particularly high expression in the intestine, uterus, ovaries and testes. Transcript levels were detected throughout all developmental stages in both ivermectin-susceptible and -resistant strains. To investigate its association with ivermectin resistance, worms were exposed to the EC₅₀ of ivermectin, which elicited a plastic expression response of \u003cem\u003eHc-NHR-49\u003c/em\u003e. RNAi-mediated knockdown of Hc-NHR-49 increased ivermectin susceptibility in resistant parasites.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCollectively, our results suggest that \u003cem\u003eHc-NHR-49\u003c/em\u003e is implicated in ivermectin resistance in \u003cem\u003eH. contortus\u003c/em\u003e. These findings contribute to a deeper understanding of resistance mechanisms and could inform the future development of alternative control measures.\u003c/p\u003e","manuscriptTitle":"Transcriptional profiling and functional characterization of the Hc-NHR-49 gene in ivermectin resistance of Haemonchus contortus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-14 18:09:15","doi":"10.21203/rs.3.rs-8472203/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-02T15:28:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-15T05:31:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291719328245259629507420310519125466124","date":"2026-01-30T00:29:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167108251981178591853964831936672162917","date":"2026-01-28T10:42:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158259875359479715269335950498601834649","date":"2026-01-13T21:15:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-09T12:33:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-31T14:06:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-31T06:32:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-12-29T10:34:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4c12ccd5-b28c-4313-9088-341512b45808","owner":[],"postedDate":"January 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T02:24:55+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-14 18:09:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8472203","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8472203","identity":"rs-8472203","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}