Tryptic enzyme activity of five individual fish larvae per tank
and treatment was assayed following a fluorescence-method
described by Ueberschar€ [39] modified for microtiter plates. In
brief, 250 mL substrate (Na-benzoyl-L-arginin-4-
methylcoumarinyl-7-amid, Bachem AG, Switzerland) were added
to 50 mL homogenate of the individual fish larva in a 96-well-plate.
After mixing and 20 min incubation at room temperature, the
relative fluorescence enhancement was recorded every 2 min for
12 min using a microtiter fluorescence reader (Fluoroskan Ascent,
Labsystems Thermo). The tryptic enzyme activity is given as an
equivalent of hydrolysed substrate per time unit and is normalized
against larval area [mm2
] (nmol hydrolysed substrate/min*larva).