A fluorescent-sensitive assay demonstrated the exhaustive detection of proteinase activities in the dorsal skin of the European eel. Two distinct skin extracts were prepared from skin mucus and epidermal cell layers with no mutual contamination, so that the latter extract contained significant susceptibility of all tested substrates. Optimum hydrolysis pH's for susceptible substrates were found in acidic and neutral ranges, and optimum hydrolysis temperatures for the same substrates fell mainly in the 40 degrees-50 degrees C range. In addition, diverse inhibitory influences on these hydrolyses were prompted by several proteinase inhibitors and metal chlorides, and some other reagents specifically affected the individual hydrolysis; among them, the inhibitions of all activities by p-tosyl-L-phenylalanyl-chloromethylketone CdCl2, CuCl2, HgCl2, and ZnCl2 were remarkable. Antipain, iodoacetamide, and CoCl2 induced a severe inhibition of all except three activities, whereas N-ethylmaleimide markedly inhibited only these three activities. These findings suggest that epidermal cell layers of the European eel retain a party of proteolytic enzymes, and this party is judged from their exhibiting specificities to be composed of four distinct proteinases, presumably cathepsins L and B, a serine proteinase, and an aminopeptidase.

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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 dif ferent 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 meth ods 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.

ABSTRACT-The present study suggests that the myxosporean emaciation disease by enteric infection of Enteromyxum leei disrupts intestinal water uptake of tiger puffer Takifugu rubripes. This idea is based on a significant negative correlation between plasma chloride concentration and condition factor in diseased fish, and significantly higher osmolarity of plasma and major ion concentrations of the intestinal fluid in infected fish than in healthy control fish. Additionally, surgical ligation of the junction between the stomach and the intestine of healthy fish resulted in a 24% drop of body weight within 50 h, and significantly lower water content of the white muscle (operated fish 74 ± 0.5%; sham-operated fish 82 ± 1%). However, in vitro water uptake by isolated intestine sacs was not significantly different between the control and infected fish. Meanwhile, hepatic function appeared to be impaired as evidenced by the significantly lower hepato-somatic index (control fish 8.7 ± 0.8%; infected fish 2.7 ± 0.8%). Plasma activities of LDH, AST and ALT were all significantly lower in the infected fish. We propose that rapid loss of body weight of infected tiger puffer is mainly due to osmoregulatory failure but probably malnutrition is also involved in the pathogenesis.