Ivermectin is an important broad-spectrum anthelmintic used to treat nematode parasites including gastro-intestinal infections of humans and animals. The mode of action for Ivermectin is understood to involve activation of inhibitory glutamate-gated chloride channels (GluCls). Ivermectin has also been reported to inhibit the release of extracellular vesicles (EVs). We found that EVs are released from the whole intestine of the gastro-intestinal parasite, Ascaris suum. Proteomic analysis identified 1,574 proteins within these intestinal EVs, including 96 nematode proteins with putative immune-associated functions based on homology to proteins involved in host immune processes and 130 proteins with predicted digestive functions. Comparative analysis following ivermectin exposure revealed 38 differentially abundant proteins that included the putative immune-related proteins: transthyretin-like proteins, a small heat-shock antigen, a phospholipase A2, and the NF-κB subunit p105. Thus, ivermectin modulated the potential immune-related cargo of intestinal EVs. The ivermectin inhibition of intestinal EV release was concentration-dependent with an IC₅₀ of 64 nM. We also identified the expression of GluCl subunit receptor genes in the Ascaris intestine. The potent inhibitory effect of ivermectin on the release of these EVs from the nematode intestine and the expression of GluCl channel subunits sheds further light on the site and mechanisms of action of this important anthelmintic.

We show the dataset’s for the figures 2,3,4,5,6,7, supplementary file S1 NS s3.

2.1 Collection and maintenance of A. suum

Adult female A. suum worms were collected from the JBS Swift and Co. pork processing plant, Marshalltown, Iowa. Worms were maintained in Ascaris Ringers Solution (ARS: 13 mM NaCl, 9 mM CaCl₂, 7 mM MgCl₂, 12 mM C₄H₁₁NO₃, Tris, 99 mM NaC₂H₃O₂, 19 mM KCl, and 5 mM glucose pH 7.8) at 32°C for 24 h to allow for acclimatization before use in experiments. The worms were used the following day. All the worms were examined at the start of each experimental day and discarded if they were damaged or immotile.

2.2 Collection of Extracellular Vesicles (EVs) from A. suum Intestinal Sections

The whole intestine of adult A. suum was extracted from the parasite by dissecting the parasite from the base of the pharynx to the anal pore (Harpur, 1977). The intestine was removed from the body using fine forceps, avoiding other tissues including the reproductive organs and muscle bags. The intestines were either processed whole or dissected into three anatomical regions: 1) the anterior region, which is primarily involved in digestion stretching from the base of the pharynx to the vulvar pore; 2) the middle region which is associated with nutrient absorption and extends from the vulvar pore to the ovarian tissue; and 3) the posterior region from the ovarian tissue to the anal pore (Fig 1A) (Harpur, 1977). The intestinal pieces were opened longitudinally, exposing the lumen, and thoroughly washed with Ascaris Perienteric Fluid (APF: 23 mM NaCl, 110 mM sodium acetate, 24 mM KCl, 1 mM CaCl₂, 5 mM MgCl₂, 5 mM HEPES, and 11 mM D-glucose; pH 7.6) to remove residual luminal contents and debris.

Intestinal sections were weighed and transferred into 15 mL tubes containing 10 mL APF supplemented with 0.01% dimethyl sulfoxide (DMSO) and were incubated at 37 °C for 4 hrs. to promote EV secretion. Following incubation, the intestinal sections were removed, and the conditioned APF solutions were collected for EV isolation and characterization. A total of fourteen independent worms as biological experiments were performed for all sections tested. For NTA analysis, conditioned APF solution from whole intestine, anterior, middle, and posterior intestinal sections were diluted 500-fold in filtered dPBS prior to particle quantification.

2.3 Measuring the Effects of Ivermectin on Intestinal EV Secretion

To assess the effect of ivermectin on EV secretion from the intestine, the anterior, middle, and posterior intestinal sections were divided equally into two halves (anterior and posterior), allowing for paired comparisons within the same worm. For half of the experiments, the anterior half of the intestinal section was incubated in 10 mL APF containing 0.01% DMSO, while the posterior half was incubated in 10 mL APF containing 1 µM ivermectin. In the remaining experiments, the treatment order was reversed, with the anterior half of the intestinal section being treated with 1 µM ivermectin and the posterior half treated in 0.01% DMSO. All samples were incubated at 37 °C for 4 hrs. A total of fourteen independent biological experiments were performed for all sections tested. For NTA analysis, conditioned APF solutions from ivermectin and DMSO treated anterior, middle, and posterior intestinal sections were diluted 100-fold in filtered dPBS prior to particle quantification.
 
For dose-response experiments the anterior sections of A. suum intestines were divided into six 1 cm pieces. For all experiments, samples were incubated in either 10 μM, 1 μM, 100 nM, 10 nM, 1 nM, and 100 pM ivermectin, at 37°C for 4 hrs. For three experiments, the samples were treated with the piece nearer the mouth being exposed to the highest ivermectin concentration and the piece near the vulvar pore being treated with the lowest concentration. To control positional bias in the remaining three experiments, the assignment was reversed with the piece near the vulvar pore being exposed to the highest concentration of ivermectin and the piece near the mouth being exposed to the lowest. For control experiments the anterior intestines of six individual A. suum were dissected into six 1 cm pieces and incubated in APF containing 0.01% DMSO at 37 °C for 4 hrs. At the end of incubation, all solutions were collected for EV isolation and quantification. For NTA analysis in the ivermectin dose-response experiments, conditioned APF solutions from anterior intestinal sections were diluted 100-fold in filtered dPBS prior to particle quantification.

2.4 EV isolation, quantification, and characterization

After incubation, conditioned solutions were passed through a 0.2 µm PVDF syringe filter (GE Healthcare, Chicago, IL) and ultracentrifuged at 120,000 × g for 90 mins at 4°C. Supernatants were decanted, leaving approximately 1.5 mL above the pellet to avoid disturbance. The EVs contained in the pellet were purified by size-exclusion chromatography (SmartSEC™ single columns), and the EV-containing fractions were concentrated by ultracentrifugation at 186,000 × g for 2 hrs. at 4°C. The pellets were resuspended in 500 µL of sterile-filtered dPBS (Thermo Fisher Scientific). Samples were diluted in filtered dPBS to achieve particle counts (20-120 particles per frame). Dilution factors were optimized for each experiment and are indicated in the corresponding sections, 2.2 and 2.3. For each sample, videos of 5 x 60 sec were recorded at a camera level of 12, and particles were detected using a detection threshold of five. The EV concentration and size distributions were determined by nanoparticle tracking analysis (NTA; Nano-Sight LM10, Malvern Instruments, Malvern, UK)

2.5 EV visualization by Transmission Electron Microscopy (TEM)

Aliquots of purified EVs were fixed in 3% glutaraldehyde + 1% paraformaldehyde in 0.1 M cacodylate buffer. 200-mesh copper grids with carbon film (EMS) were glow-discharged (Pelco easiGlow, Ted Pella) to render a hydrophilic surface. 2 µL of fixed EV suspension were applied to each grid for 30s, excess wicked with filter paper, and immediately negative-stained with 2% (w/v) uranyl acetate (2 µL, 30s), then wicked and air-dried. Grids were examined on a JEOL 2100 transmission electron microscope operated at 200 kV and imaged with a Gatan OneView camera at the Iowa State University Light and Electron Microscopy Facility.

2.6 Proteomic Analysis of EV Contents

EV protein lysates were prepared from EV pellets prior to LC–MS/MS proteomic analysis. EV pellets were kept on ice and resuspended in 30 µL of 8 M urea prepared in 50 mM ammonium bicarbonate buffer (ABC, pH 8.5). The urea–ABC lysis buffer was prepared at room temperature without heating and deionized using AG 501-X8 resin before use to reduce ionic contaminants and minimize urea-derived carbamylation. EV pellets were lysed by repeated pipetting, vortexing, and incubation at room temperature. Specifically, each sample was pipetted up and down approximately 100 times, vortexed for 1 min, incubated at room temperature for 10 min, and vortexed again for 30 s. This cycle was repeated twice to promote complete solubilization of EV-associated proteins. The lysates were then centrifuged at 12,000–16,000 × g for 10 min at 4 °C to remove insoluble debris. The resulting supernatant was collected as the soluble EV protein lysate for downstream proteomic analysis. When protein quantification was required, aliquots were diluted to reduce the urea concentration before measurement or analyzed using a urea-compatible protein assay (Wiśniewski et al., 2009, Rontogianni et al., 2019). Samples were maintained on ice or at 4 °C for short-term handling and were stored at −80 °C if same-day processing was not possible, with repeated freeze–thaw cycles avoided.

2.7 Protein digestion and LC–MS/MS analysis

Crude EV protein extracts were processed for bottom-up proteomic analysis by in-solution enzymatic digestion. Briefly, protein extracts were reduced with dithiothreitol (DTT), and cysteine residues were alkylated with iodoacetamide prior to enzymatic digestion. Samples were then digested overnight with trypsin/Lys-C. Digestion was stopped by the addition of formic acid, and the samples were dried using a SpeedVac concentrator. Peptides were desalted using C18 columns (BioPureSPN Plate, HHNFR S18V; Nest Group) and dried again in a SpeedVac. Peptide concentration was determined using a bicinchoninic acid assay kit (BCA-1; Sigma-Aldrich). Peptide Retention Time Calibration standard mixture (PRTC; Pierce, part No. 88320) was spiked into each sample as an internal control for LC–MS/MS performance monitoring. Samples were normalized to 200 ng/µL peptide and 50 fmol/µL PRTC, and 2 µL of each sample was injected for LC–MS/MS analysis.

Peptides were separated by liquid chromatography using a Thermo Scientific Vanquish Neo UHPLC system and analyzed by tandem mass spectrometry on a Thermo Scientific Orbitrap Astral mass spectrometer equipped with a Thermo Scientific EASY-Spray ion source and column. The resulting intact and fragmentation pattern is compared to a theoretical fragmentation pattern using CHIMERYS. CHIMERYS was used to identify and quantify peptides from complex tandem mass spectra, including spectra containing co-fragmented peptide ions. Protein identification and quantification were performed based on peptide-spectrum matching and comparison of the observed MS/MS fragmentation patterns with theoretical fragmentation patterns generated from the protein database. The complete proteomic dataset, including protein identifications, peptide counts, and grouped abundance values for all DMSO and ivermectin treated replicates, is provided in Supplementary Data File S1.

2.8 Functional classification of extracellular vesicle proteins

To characterize the functional composition of the intestinal extracellular vesicle proteome, identified Ascaris suum proteins were assigned to functional categories using a priority-based scheme that combined UniProtKB (release 2026_02; accessed June 2026)annotation with manual curation. For each protein, the UniProtKB protein name and Gene Ontology (GO) biological process terms were retrieved and grouped according to their GO biological process terms. All remaining proteins lacking GO annotation were classified separately. The curated keyword lists were compiled from protein families previously reported in nematode and helminth secretome and extracellular vesicle studies, and all family assignments were manually reviewed. A. suum is not represented in standard GO-slim enrichment databases (e.g., PANTHER), so a curated approach was applied in place of automated analysis. Category and sub-family counts were visualized as pie and donut charts.

2.9 Measurement of differential abundance analysis and visualization

Extracellular vesicle proteins were performed using the grouped protein abundances generated by Proteome Discoverer, comprising five ivermectin-treated and five DMSO-treated (vehicle control) replicates, with all abundances normalized to the PRTC spike-in standard. 'Master Protein' (Any UniProt-mapped A. suum) were retained, and contaminant entries (Cont_) and PRTC peptides were removed. All Ascaris suum proteins were mapped to UniProt identifiers. For each protein, we measured the abundance values in each treatment group, and the valid values were checked. Proteins below two valid abundance values in either treatment group (only present in 1 worm) were excluded from the differential-abundance test as a reliable estimation was not possible. The grouped protein abundances from five ivermectin-treated and five DMSO-treated replicates were log₂-transformed. For each protein, the log₂ fold change was calculated as the difference between the mean log₂ abundance of the ivermectin groups and that of the DMSO groups (mean log₂ DMSO, mean log₂ IVM; log₂ FC = mean log₂ IVM - mean log₂ DMSO). To determine significance the five IVM log₂ vs five DMSO log₂ were assessed using Welch's t-test (unequal variance), and p-values were adjusted for multiple comparisons using the Benjamini–Hochberg false discovery rate (FDR). Proteins with p < 0.05 and log₂ FC > 1 were considered candidate differentially abundant proteins. For heatmap visualization, abundance values were converted to z-scores across the ten samples for each protein [z = (log₂ value - mean)/SD, across the 10 samples (DMSO & IVM)]. Volcano plots were generated by plotting each protein log₂ FC (x) vs -log₁₀(p) (y).

2.10 A. suum cDNA synthesis and RT-PCR detection of Glutamate-gated Chloride (GluCl) channels

A 3 cm segment of the anterior body of adult female A. suum was excised and opened longitudinally. The intestine was separated from the body wall using two fine forceps and each tissue was transferred to 1.5 mL RNase-free tubes. Tissues were snap-frozen in liquid nitrogen and stored at −80°C until processing. RNA was isolated from frozen tissues that were homogenized separately in 1 mL TRIzol™ Reagent (Life Technologies, USA) using a mortar and pestle according to the manufacturer’s protocol. cDNA was synthesized from one microgram (1 μg) RNA per sample using SuperScript IV VILO™ Master Mix (Life Technologies, USA). Paired intestine and body wall tissues were collected from five individual adult female worms for RT-PCR analysis.

RT-PCR was performed to detect Asu-glc-2, Asu-glc-3, Asu-glc-4, and Asu-avr-14 (Supplementary Table 2) in the cDNA pool of the intestine and body wall using primers targeting the coding region of each gene (Supplementary Table 1). The reference gene Asu-gapdh served as the positive control. Negative controls included enzyme, water, and both forward and reverse primers for the target gene with no cDNA template. RT-PCR detection was performed on cDNA derived from all five individual worms described above. The cycling conditions for PCR were an initial denaturation for 2 min at 95°C, followed by 35 cycles at 95°C for 30s, 60°C for 35s, 72°C for 45s, followed by a final extension period at 72°C for 10 mins using GoTaq® G2 Hot Start Green Master Mix (Promega, USA). The PCR products of each gene were then separated on an individual 2% Agarose gels containing SYBR® Safe DNA Gel Stain (ThermoFisher Scientific), for 60 mins at 100 V, followed by visualization under UV light to confirm the presence of the genes. All photographs were acquired using Visionworks™ software (Analytik Jena) with an exposure setting of 3s per 1 frame.

RT-PCR was performed to generate full length transcripts of Asu-avr-14 from the cDNA of the parasite intestine using primers that targeted the start and end codon (Supplementary Table 1). The reference gene Asu-gapdh served as the positive control. Negative controls included enzyme, water, and both forward and reverse primers for Asu-avr-14 with no cDNA template. Amplification was performed with Platinum SuperFi™ II Green PCR Master Mix (Thermo Fisher Scientific) under the following conditions: 98 °C 30s; 35 cycles at 98 °C 30 s, 59 °C for 20 s, 72 °C for 40 s and a final extension at 72 °C for 10 mins. Products were identified on a 1% agarose gel containing SYBR® Safe (Thermo Fisher Scientific) at 100 V for 60 mins, visualized under UV illumination, and imaged with VisionWorks™ (Analytik Jena; 3 s exposure). Target bands were excised and purified with the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) per the manufacturer’s instructions. Purified amplicons were submitted to the Iowa State University DNA Sequencing Facility for Sanger sequencing (bidirectional reads were achieved using the same forward and reverse primers).

2.11 Statistical analysis

Statistical analyses were performed using Prism version 10.0 (GraphPad Software, La Jolla, CA, USA). EV concentrations were normalized to tissue wet weight (mg) for analysis. Differences in EV secretion among intestinal regions were analyzed using one-way ANOVA followed by a post hoc Šídák multiple-comparisons test. The effect of ivermectin on regional EV release was analyzed using an unpaired t-test, with a P value < 0.05 being considered statistically significant for all analyses. Dose–response relationships were analyzed using nonlinear regression with a log (inhibitor) versus response–variable slope model, and ivermectin concentrations were log₁₀-transformed prior to analysis. A two-way ANOVA model was used to compare the means of EV size profiles following drug treatment, and statistical significance was determined using a post hoc Šídák multiple-comparisons test (P < 0.05 was considered significant). To account for the wide dynamic range of EV concentration values (particles mL^-1 mg^-1) across size subsets, a constant value of 1 was added to all EV concentration values (8 size subsets x 14 DMSO and ivermectin treated replicates per region) prior to log transformation, to avoid undefined values arising from zero counts in the log transformation. The adjusted values were them log₁₀-transformed. A two-way ANOVA model was applied to the log₁₀-transformed data to compare EV size profiles following drug treatment, and statistical significance was determined using a post hoc Šídák multiple-comparisons test (P < 0.05 was considered significant).

Proteomic data processing and statistical analyses were performed in Python using the pandas, NumPy, and SciPy packages, and figures were generated in R (version 4.5.2). Differential protein abundance between ivermectin and DMSO treated intestinal EVs was assessed by Welch's t-test on log₂-transformed abundances to stabilize the variance, approximate a normal distribution and not assume equal variances between groups (n = 5 replicates). P values were corrected for multiple testing using the Benjamini–Hochberg false-discovery-rate (FDR) procedure. Proteins with P < 0.05 and log₂ fold change >1 were considered differentially abundant. Given the sample size, these proteins are reported as candidate differentially abundant proteins for functional interpretation. To ensure reproducibility, experiments were repeated independently, and details including the number of adult female worms used, intestinal preparations, drug concentrations, and treatment durations (IVM and DMSO) are provided in the corresponding figure legends.