We examined whether infection by the monogenean Heterobothrium okamotoi induces production of specific antibodies against oncomiracidia and their cilia, larvae on the gills, and adults on the branchial cavity wall of tiger puffer Takifugu rubripes. We also investigated whether specific antibody production participates in acquired protection against H. okamotoi. Sera from persistently infected fish immobilized H. okamotoi oncomiracidia 89 days after exposure and antibody levels (measured by enzyme-linked immunosorbent assays) in the sera against oncomiracidia and their cilia increased compared with sera from control (naïve) fish. Antibody levels in these sera against the larvae and adult stages did not increase. The number of H. okamotoi on persistently infected fish was significantly lower than for control fish (P<0·05) when persistently infected fish and control fish were exposed to oncomiracidia in the same tank. Thus tiger puffer produced specific antibodies against oncomiracidia and their cilia, and acquired partial protection against H. okamotoi. Intraperitoneal injection of proteins of sonicated oncomiracidia or their cilia with an adjuvant also produced oncomiracidium agglutination antibodies in sera from tiger puffer; the antibody levels in these sera against oncomiracidia and their cilia increased compared with sera from control fish (injection of BSA with an adjuvant) at 14, 44, and 75 days after the booster immunization. However, in a parasite challenge at 54–58 days after the booster immunization, the infection levels of fish immunized with parasites of sonicated oncomiracidia or their cilia were the same as the control fish. Western blot showed that sera from persistently infected fish and fish immunized with sonicated oncomiracidia or their cilia recognized similar antigenic bands, suggesting that tiger puffer tends to react against these antigens compared with other antigens. These results indicated that specific antibodies against these cilia and oncomiracidia induced by i.p. injection do not prevent H. okamotoi infection.

We examined a host-finding factor of the monogenean Heterobothrium okamotoi oncomiracidia to develop an alternative prophylaxis. H. okamotoi oncomiracidia attached preferentially to gill filaments and skin mucus from the tiger puffer Takifugu rubripes compared with corresponding material from other tested fishes (amber jack Seriola dumerili, red sea bream Pagrus major, Japanese flounder Paralichthys olivaceus and spotted halibut Verasper variegatus). The body mucus pH of the tiger puffer was 6·40±0·09 (mean±S.D.), whereas that for the other tested fishes was 7·2–7·4. To find if this difference in pH could account for the specific targeting of tiger puffer by H. okamotoi oncomiracidia, the attachment response of the oncomiracidia to pieces of agar buffered at various pH between 6·0 and 7·4 was examined. The number of attaching oncomiracidia was maximal at pH 6·4. We produced gynogenetic tiger puffers from a single female. These gynogenetic individuals showed a variety of body mucus pH and they were exposed to the oncomiracidia. Thirteen days after exposure, more young H. okamotoi were found on the gills of gynogenetic tiger puffer with mucus at pH 6·3–6·6, than on gills of fish with mucus at pH 6·0–6·3 and 6·6–7·2. H. okamotoi exploits the body mucus pH to identify the host. The simplicity of pH as a lure may lead to development of a simple and economical method to control H. okamotoi outbreaks.