J. Limnol., 68(2): 409-412, 2009 DOI: 10.3274/JL09-68-2-n1 Differentiation between activity of digestive enzymes of Brachionus calyciflorus and extracellular enzymes of its epizooic bacteria Martina ŠTROJSOVÁ 1,2) * and Wilko H. AHLRICHS 3) 1) University of South Bohemia, Faculty of Science, Branišovská 31, CZ–370 05 České Budějovice, Czech Republic 2) Biology Centre AS CR, Institute of Hydrobiology, Na Sádkách 7, CZ–370 05 České Budějovice, Czech Republic 3) Carl von Ossietzky Universität, Department of Biology and Environmental Sciences, D-26111 Oldenburg, Germany *e-mail corresponding author: martina.strojsova@seznam.cz ABSTRACT The rotifer Brachionus calyciflorus was examined by scanning electron microscopy (SEM) for surface-attached, i.e. epizootic, bacteria to ascertain their specific localization and thus find out if we could discern between rotifer and bacterial enzyme activity. The lorica of B. calyciflorus was colonized by one distinct type of bacteria, which originated from the algal culture used for rotifer feeding. The corona, posterior epidermis and foot of all inspected individuals were always without attached bacteria. The density of the attached bacteria was higher with the increasing age of B. calyciflorus: while young individuals were colonized by ~ tens of bacterial cells, older ones had on average hundreds to thousands of attached bacteria. We hypothesize that epizooic bacteria may produce the ectoenzymes phosphatases and β-N-acetylhexosaminidases on the lorica, but not on the corona of B. calyciflorus. Since enzyme activities of epizooic bacteria may influence the values and interpretation of bulk rotifer enzyme activities, we should take the bacterial contribution into account. Key words: rotifers, phosphatase, β-N-acetylhexosaminidase, enzyme localization 1. INTRODUCTION Brachionus calyciflorus is a common species in freshwater ponds, lakes and reservoirs, almost cosmo- politan and often cultured for laboratory experiments. In our preceding study, B. calyciflorus was used in investi- gating digestive enzymes (Štrojsová & Vrba 2007). Hydrolytic enzymes involved in rotifer digestive proc- esses could be detected and/or measured either in rotifer homogenates or directly at the site of enzyme action in intact rotifers. Because non-axenic rotifer cultures are commonly used, the contribution of enzyme activities of organisms other than B. calyciflorus has to be considered. Localizations of enzyme activities of B. calyciflorus were previously studied by the Fluorescently Labelled Enzyme Activity (FLEA) technique (Štrojsová & Vrba 2007). The FLEA technique, together with image analy- sis software, can also be used for quantification of enzyme activity (Nedoma et al. 2003; Štrojsová & Vrba 2008). The fluorogenic ELF ® 97 substrates are soluble in water and could be ingested by the rotifers when feed- ing. After enzymatic hydrolysis, the non-fluorescent substrate turns into insoluble fluorescent ELF alcohol that precipitates and tags the enzyme activity outside or inside the rotifer body. The activity of phosphatase and β-N-acetylhexosaminidase was observed mainly in the digestive tract, but was also observed at the corona and on the lorica of B. calyciflorus (Štrojsová & Vrba 2007). While it is possible to hypothesize a digestive function of enzyme activity, its function within the corona and lorica is still unknown. In our previous study (Štrojsová & Vrba 2007), epi- zooic bacteria were not observed on the rotifer body stained with DAPI (Porter & Feig 1980); however, epi- zooic bacteria were commonly observed on the epider- mis of B. plicatilis (Kleinow 1993). Because of possible overlap of the fluorescence of ELF alcohol and DAPI, we were not completely convinced that the enzymatic activities were exclusively of rotifer origin (Štrojsová & Vrba 2007). To solve this problem, we used a more sen- sitive method, scanning electron microscopy (SEM), to detect possible epizooic bacteria on the surface of B. calyciflorus. We inspected the same culture of B. caly- ciflorus which we used to study rotifer digestive enzymes. 2. METHODS Brachionus calyciflorus Pallas, isolated from Lake Constance, was obtained from the Leibniz Institute of Freshwater Ecology and Inland Fisheries (Berlin, Ger- many) and cultured in 6-mL chambers of 12-well microtiterplates in Z/4 medium (Zehnder & Gorham 1960). Stock cultures were diluted weekly (1:2; cul- ture/medium) and incubated at 19 ± 1 °C under a 12:12- h light/dark regime. Rotifers were fed on Chlorella kessleri Fott et Novakova (Culture Collection of Algal Laboratory, Institute of Botany AS CR, Třeboň, Czech Republic) once a week. B. calyciflorus was prepared for SEM according to Riemann et al. (2008). The rotifers were narcotized by water enriched with CO 2 , then killed by a small amount of 1% osmiumtetroxide (1% OsO 4 in 0.1 M sodium cacodylate buffer). Finally, the rotifers were washed in J.L. NOTES