Ethics statement

The study protocol was revised and approved by the Animal Care and Use Committee (CIEA, in Spanish) at Universidad Peruana Cayetano Heredia (CIEA certificates 040-09-22, R 040-09-23, and R 040-09-24, valid until August 2025). The CIEA states details for the proper housing, feeding, and handling of animals used in research, including euthanasia. This committee is registered with the Office of Laboratory Animal Welfare of the Department of Health and Human Services of the National Institutes of Health (NIH—USA) with Assurance Number F16-00076 (A5146-01), valid until August 31, 2026.

Collection of T. solium cysts from pig muscle

Taenia solium cysts (n = 500) were collected under aseptic conditions at Universidad Peruana Cayetano Heredia’s Global Health Center in Tumbes, Peru. The parasites were excised from the muscle tissue of a naturally infected pig carcass using sterile scalpels, ensuring that the surrounding capsule of inflammatory infiltrate and collagen was removed entirely. Upon collection, cysts were washed three times in sterile 50-mL Falcon tubes with cold transport solution (1X PBS, pH 7.2, with Gibco^TM Antibiotic-Antimycotic to the following final concentrations: 100 units/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B). Each wash involved gently inverting the tube several times over a period of 3 minutes; the solution was replaced between washes. Cysts were transferred to sterile 50-mL Falcon tubes (up to 100 cysts per tube) with at least twice their volume of fresh transport solution and packed in an insulating container that was kept below 12–15 degrees until its arrival by air freight to the university laboratories in Lima.

In vitro scolex evagination and morphology of activated parasites

Cysts arrived at the laboratory approximately 16 hours after collection and were washed three times with transport solution like before, maintaining an aseptic environment. After shipping and washing, most cysts maintained their characteristics: intact, white, or translucent oblong-shaped vesicles without atypical coloration, filled with a clear fluid (Fig. 1b). Cysts were then distributed for different purposes; 84 were used for the present study.

Twelve cysts were placed each in 1 mL of RNAlater® solution (R-0901, Sigma Aldrich) and stored at −20 °C until RNA extraction. These ex vivo cysts were defined as non-cultured (NC).

For culture, washed cysts were transferred to culture plates with sterile Pasteur pipettes, working in a laminar flow hood. Each one of 72 cysts was incubated in 1.5 ml filtered sterilized RPMI 1640 medium (pH 7.4), supplemented with 2 g/mL NaHCO₃ (Merck®, Burlington, MA), 1.6 µM β-mercaptoethanol, and Gibco™ Antibiotic-Antimycotic in the concentrations described above. One cyst per well was placed in three Costar® 24-well clear Tissue Culture-treated surface plates (Corning®, Corning, NY, USA) at 37 °C and 5% CO₂ in a sterile incubator (VWR Symphony Incubator) for up to five days. The medium of 36 of the 72 cysts included 0.1% taurocholic acid (TA) (Sigma, St. Louis, MO, USA). Therefore, half of the samples were TA+ and half TA-. The medium was replaced every 24 hours. Cysts were collected in three distinct moments: cysts with non-evaginated scolex, after 6 hours in vitro, TA+ and TA- (defined as “pre-evagination”: PRE TA+ and PRE TA-); cysts with recently exposed scolex, approximately after 24–48 hours in vitro (defined as “recent evagination”: EV TA+ and EV TA-); and cysts having fully evaginated the scolex, already showing some proglottids, after 120 hours in vitro (defined as “post-evagination”: POST TA+ and POST TA-). After culture, all parasites were washed with 1X PBS to remove residual media, and preserved at −20 °C in 1 mL RNAlater® solution until RNA extraction. Evagination curves were made along the 120 hours in vitro.

RNA isolation

RNA was extracted from 12 samples (cysts) of each of the seven groups (NC, PRE TA+, PRE TA-, EV TA+, EV TA-, POST TA+, POST TA-; n = 84 cysts) using the Quick-RNA Miniprep Plus Kit (Zymo Research, R1058). Each preserved cyst was placed on a sterile Petri dish, and its vesicle was carefully removed with a sterile scalpel, leaving only the parenchymatous portion (scolex, neck, and eventual proglottids). Samples were independently washed thrice with molecular biology grade water (Molecular grade; Fisher Scientific), then embedded in 600 µL of DNA/RNA Shield (Zymo Research, R1058), and admixed with a sterile tissue homogenizer tip (Omni International, Kennesaw, GA) on ice, followed immediately by a 30-min treatment with 60 mg of Proteinase K (Zymo Research, D3001-2-60). RNA was isolated following the manufacturer’s protocol, with an extra washing step with 400 µL of RNA Wash Buffer (Zymo Research, R1058) before final RNA isolation. The protocol included DNAse I treatment (Zymo Research, E1009-A (250 U)). As recommended by the manufacturer, a mix of 5 µL of DNase I (1 U/µL) and 75 µL of DNA Digestion Buffer (Zymo Research, R1058) was added directly to the silica column matrix (Zymo-Spin™ IIICG Columns) containing processed samples, for a 15-min incubation at room temperature (20–30 °C). RNA yield was measured by spectrophotometry with NanoDrop® (Thermo Fisher Scientific, Waltham, MA, USA), and approximately 300 ng of RNA were loaded into a 1.5% agarose/1% bleach gel to test its integrity by electrophoresis (35 minutes at 100 V)²⁷.

RNA-Seq library construction and sequencing

We constructed 21 libraries, three for each of the seven groups (NC, PRE TA+, PRE TA-, EV TA+, EV TA-, POST TA+, POST TA-). Each library was made from RNA from four cysts, pooling 125 ng of independently extracted RNA for a final 500 ng/library. Following the manufacturer’s instructions, the Illumina® Stranded mRNA Prep Ligation Kit (Illumina, 20040532) was used to construct libraries indexed with the IDT for Illumina RNA Unique dual (UD) Indexes. Briefly, Oligo(dT) RNA purification magnetic beads (RPBX, Illumina, 20040532) allowed for polyA-RNA selection and excluded ribosomal RNA. Fragmentation of purified mRNA, cDNA synthesis of the first and second strands, and ligation of the Illumina RNA UD Indexes Set A followed. The libraries were amplified by 13 PCR cycles and quantified using the Qubit™ dsDNA High Sensitivity (HS) Assay Kit (Thermo Fisher Scientific, Q32851). The library fragment sizes and their integrities were verified on 2% agarose gel electrophoresis.

Each library was diluted to a starting concentration of 1 nM, and the 21 libraries were combined using 10 μL of each diluted library. The combined libraries were denatured and diluted to a final concentration of 1.4 pM before sequencing. Libraries were sequenced for 75 cycles in paired-end mode with a high output kit on the Illumina NextSeq 500 platform (Illumina, San Diego, CA, USA) according to the Illumina protocol. FASTQ files were generated using the bcl2fastq software (Bcl2Fastq; Illumina). FASTQ data was deposited in GEO (GSE288552)²³.

RNA-seq data analysis

Quality control of the reads was assessed with FastQC (version 0.12.0)²⁸. Removal of Illumina adapter sequences and trimming of the reads with a phred + 33 quality score below 20 was done using BBDuk (BBMap version 38.90)²⁹. Reads were mapped to the T. solium genome (assembly GCA_001870725.1)¹³ using the default settings of RSubread (version 2.17.4)³⁰. Gene abundance was calculated with GenomicFeatures (v. 1.52.1)³¹ using predicted gene annotations available in the WormBase ParaSite database (version WBPS18)^13,32.

Variance-stabilized counts were calculated using a generalized linear model that assumes a negative binomial distribution of count data, implemented by DESeq 2 (v.1.40.2)³³, and were used for a principal component analysis (PCA) using the 500 genes with the highest variance. Additionally, hierarchical clustering was performed across all sample counts by calculating a Euclidean distance-based matrix.

Functional annotation of the T. solium genome

We retrieved full-length T. solium transcript sequences from the WormBase Parasite database (PRJNA170813)^13,32. After importing the sequences into Blast2GO (OmicsBox suite)²⁴, we replaced duplicate IDs and added descriptions. A DIAMOND blastx search was conducted against the NR database with an E-value of 1 × 10⁻⁵ and a taxonomy filter for Platyhelminthes (6157), Trematoda (6178), Cestoda (6199), and Nematoda (6231).

We used InterProScan³⁴ version 5.69–101.0 (July 2024) to identify conserved domains, motifs, and signal peptides, applying various databases; see Table 1. High-confidence functional annotations were assigned using an E-value threshold of 1 × 10⁻⁶, an annotation cutoff of 55, and a GO weight of 5. Additionally, we integrated functional annotations from eggNOG 5³⁵ to enhance annotation quality with orthologous group information using a seed ortholog E-value filter of 1 × 10⁻⁵ and a seed ortholog bit-score filter of 60.0.

Finally, we exported the annotated data into R³⁶ (version 4.4.2), creating a custom annotation package using the makeOrgPackage() function from AnnotationForge²⁵ for downstream analyses. This process provides a solid framework for exploring the functional roles of T. solium genes across different conditions.