Direct detection of digestive enzymes in planktonic rotifers using enzyme-labelled fluorescence (ELF)
M. Štrojsová A C and J. Vrba A BA University of South Bohemia, Faculty of Biological Sciences, Branišovská 31, CZ-37005 České Budějovice, Czech Republic.
B Hydrobiological Institute of the Academy of Sciences of the Czech Republic, Na Sádkách 7, CZ-37005 Č. Budějovice, Czech Republic.
C Corresponding author. Email: [email protected]
Marine and Freshwater Research 56(2) 189-195 https://doi.org/10.1071/MF04280
Submitted: 23 November 2004 Accepted: 24 February 2005 Published: 12 April 2005
Abstract
A novel enzyme-labelled-fluorescence (ELF) method was applied to natural populations of planktonic rotifers from a eutrophic reservoir. Direct visualisation of rotifers by this new method provided new information about enzymatic activities in situ, including detection and location of enzyme activities. Three fluorogenic substrates were used for the enzyme assay in concentrated (20–60×) samples of the rotifers. After a short (1–3 h) incubation in test tubes, samples were filtered and the rotifers on polycarbonate filters were examined using an epifluorescence microscope. Activity of phosphatases, β-N-acetylhexosaminidases and lipases were detected in some species that were regularly inspected during two seasons – most frequently in the stomach area, at the corona and, less often, in the mastax area. The results suggest that most of the detected enzymes are connected with the digestive tracts of rotifers. Also, autofluorescence of chlorophyll a enabled visualisation of the digestive tracts of the rotifers and provided additional information on the food (phytoplankton). Enzyme expression did not show any clear seasonal trend. Detection of specific enzymes varied considerably between species of rotifers and between individuals. This variability could be a result of change of feeding behaviour of rotifers in the concentrated samples and also could reflect individual differences among the rotifers in a population, such as feeding activity, age or life stage.
Extra keywords: β-N-acetylhexosaminidase, lipase, phosphatase.
Acknowledgments
The Grant Agency of AS CR supported this study (project A6017202 awarded to J. V.). We benefited from valuable experience and helpful advice of M. Devetter, J. Nedoma, J. Seďa and A. Štrojsová, and from their critical reading of an original draft of this paper. We further appreciated their field or laboratory assistance as well as that of Z. Prachař and M. Štojdlová. P. Znachor helped with the final set up of micrographs. Finally, we acknowledged valuable criticism of two anonymous referees.
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