2.1. Ethics statement
All experiments were conducted according to a permission obtained
by the Animal Experiments Inspectorate under the Danish
Ministry of Environment and Food (License number 2016-15-0201-
00902). Fish were euthanized before severe pathology occurred.
2.2. Fish
Zebrafish were reared in a recirculated system (Aquaschwarz,
Germany) at 27 C with a pH of 7.4 and conductivity at 550 mS. Ten
percent of the water was changed every day and the fish were fed
with live Artemia and pelleted dry feed (ZM Fish Food, England)
one to three times per day. Adults of 5 þ months were used for this
study. They were acclimatised to room temperature for two days
before the experiment started.
2.3. Parasite population
The parasites were obtained from infected Xiphophorus maculatus
in a pet shop. The parasite species verification was conducted
morphologically using a stereomicroscope identifying the ciliated
parasites with the unique horseshoe-shaped nucleus [28]. The
infected fish were euthanized in an overdose of etomidate (30 mg/
L, Sigma-Aldrich, Denmark) and left for 4 h in clean fish facility
water at room temperature, for the parasites to exit the fish. The
dead fish were removed and the parasites were left overnight for
further development into the infective stage, called theronts. The
next day the formation of theronts was verified using a dissection
microscope.