Standardized method for experimental infection of tiger puffer Takifugu rubripes with oncomiracidia of Heterobothrium okamotoi (monogenea: diclidophoridae) with some data on the oncomiracidial biology

Abstract

0-year-old tiger puffer, Takifugu rubripes, were exposed to suspensions of Heterobothrium okamotoi oncomiracidia under different conditions. A fluorescent dye, CFSE (5(6)-carboxy fluorescein diacetate, succinimidyl-ester), was used as a mean of labeling oncomiracidia to find them with a fluorescence microscope immediately after settlement on the host. The labeling procedure did not affect the infectivity of the oncomiracidium. When fish were exposed to labeled oncomiracidia for 1, 3, 5 or 10 h, the settlement rate on the gills increased with increasing exposure time up to 3 h. When similarly exposed to labeled oncomiracidia of different ages (0-4 days after hatching), infectivity of the oncomiracidium decreased rapidly with increasing parasite age, and most oncomiracidia of 2 days or older failed to infect the gills. When exposed to 29, 58 and 116 labeled oncomiracidia/ L/ fish and observed under fluorescence stereomicroscopy immediately after exposure, the settlement rates on the body surface and on the gills were not different among the 3 concentrations tested. The settlement rate on the body surface was always higher than that on the gills, irrespective of the difference in concentration. Thus, infection methods using tiger puffer and H. okamotoi oncomiracidia were standardized in terms of exposure time (3 h), parasite age (within 24 h after hatching), and parasite concentration (up to 116 oncomiracidia/ fish). CFSE-staining is suitable for the study of early stages of parasite settlement on the host fish.