Isolation of S. mansoni eggs

Eggs were isolated from the livers of hamsters six weeks after infection with 600 S. mansoni cercariae [68]. Specifically, livers were finely minced with a sterile razor blade and digested overnight at 37°C in 40 mL 1×PBS containing 2% penicillin/streptomycin/amphotericin B (1×PBS -2% PSF) and 5 mL 0.5% clostridial collagenase solution (0.025 g of collagenase (Sigma, C0130) in 5 mL of dH₂0 for immediate use). All subsequent steps proceeded at room temperature. The digested livers were centrifuged at 400 × g for 5 min and the supernatant was decanted. The resulting pellet was resuspended in 50 mL 1×PBS-2% PSF, centrifuged under the same conditions and the supernatant decanted: this step was repeated 3–5 times until the supernatant was clear. During this process, eggs settle to the bottom of the conical tube. Eggs were layered gently onto the first Percoll gradient (8 mL sterile Percoll with 32 mL 0.25 M sucrose) and centrifuged at 800 × g for 10 min to separate any remaining liver tissue debris. The supernatant was discarded with a serological pipette. Eggs were resuspended in 3 mL 1×PBS -2% PSF and gently applied to the second Percoll gradient (2.5 mL Percoll with 7.5 mL 0.25 M sucrose). Immature eggs were removed at the interface and mature eggs were collected from the bottom of the column. Immature eggs were further separated using a third Percoll gradient (6 mL of Percoll, 0.6 mL 9% saline and 3.4 mL M199 culture medium) and centrifugation for 15 min at 250 x g [55]. The mature and immature egg fractions were washed 3–5 times with M199 medium, centrifuged at 310 x g for 3 min to remove any remaining Percoll and tissue debris. Mature egg fractions typically contain less than 5% immature eggs, whereas immature egg fractions contain 5–10% mature eggs, as observed microscopically. Hatching of mature eggs in water was usually ~80% after 40 min under a bright light, thus confirming their viability. Egg size and nuclear status were checked to validate the egg developmental stage. Immature eggs were smaller, and under bright field images, there were no clear envelopes separating the embryo from the eggshell, indicating an undeveloped status [22]. In contrast, mature eggs were larger with a well-defined envelope containing a moving miracidium (S1 Table and S2A Fig). To generate dead eggs, mature eggs were treated with 1% sodium azide (Sigma S2002) for 24 h in M199 and then washed six times in the same medium. All experiments were conducted within five days of preparing eggs.