Incubation of S. solidus plerocercoids

Three-spined sticklebacks were sacrificed by pithing (destroying the brain with a needle). The fish were then individually tightly wrapped with cling film to prevent the plerocercoids from crawling out of the host's body cavity and contacting the environment during incubation. Thereafter, the infected sticklebacks were immediately placed in water heated to 40°C for 1 h (Fig. 1). At the end of incubation, the fish were quickly taken out from the water, S. solidus were removed from the host body cavity (n=3), weighed, and frozen in liquid nitrogen. Plerocercoids (n=3) taken out from the fish body cavity immediately after the sacrifice of sticklebacks kept at 22°C were used as a control group. The intensity of infection of the studied fish was one plerocercoid per fish (Table S1). This experiment was repeated twice, in 2018 and 2019.

Ten infected sticklebacks were sacrificed by pithing, while preventing pressure on the abdomen of the fish. The worms were removed from the fish body cavity and washed twice in a solution containing RPMI-1640 medium (Sigma-Aldrich) with 1% antibiotic antimycotic solution (Sigma-Aldrich). The intensity of infection of the studied fish was one plerocercoid per fish (Table S1). Next, the worms were placed in culture flasks with culture medium (RPMI-1640 medium, 0.1% antibiotic antimycotic solution and 10% glucose) and incubated in the water jacketed incubator (SHEL LAB) at 40°C (n=5) and 20-22°C (n=5) in a 5-10% CO₂ atmosphere.

Egg production was assessed twice a day visually. In the treatment group the parasites became mature within 48 h, as determined by the presence of eggs in the medium. After this, the helminths from both groups were removed from the culture flasks, washed in fresh culture medium, frozen in liquid nitrogen and stored until proteome analysis. Plerocercoids (n=3) taken out from the fish body cavity immediately after the sacrifice of sticklebacks kept at 22°C were used as a control group. The whole long-term experiment was carried out in 2019.