Grazing by amoebae causes a dramatic shift in the genetic structure of a bloom of the cyanobacterium Microcystis Jeroen Van Wichelen 1, Pieter Vanormelingen 1, Ineke van Gremberghe 1, Ann-Eline Debeer 1, Karen Soenen 1, Tine Verstraete 1, Sofie D Hondt 1, Renaat Dasseville 1, Katleen Van der Gucht 1, Jean-Pierre Descy 2, Geoffrey Codd 3 & Wim Vyverman 1 1 Research group Protistologie & Aquatische Ecologie, Ghent University, Krijgslaan 281 S8, 9 Gent, Belgium 2 Laboratory of Freshwater Ecology, University of Namur, 5 Namur, Belgium 3 Division of Environmental and Applied Biology, School of Life Sciences, University of Dundee, Dundee DD1 4HN, United Kingdom 4 Centre d ingénierie des Protéines, Institut de Chimie B6, Université de Liège, B-4 Liège, Belgique
45 4 35 3 25 2 15 1 5 Otsuka et al. 21. Int. J. Sist. Evol. Micr. 51:873-879.
Sampled blooms in Flanders About 4 % of cyanobacterial blooms contained microcystins
Leeuwenhofvijver (Drongen) 8 9 24
Tiense broeken 7 8 25
l/m² % of nursed birds in Belgian wildlife rehabilitation centers h C Environmental factors favouring avian botulism in Belgium 18 17 16 15 botulism victims amongst waterfowl 16 * average summer (May-October) temperature x 14 12 1 8 6 4 2 Climate change Evolution in average temperature in Belgium (Ukkel, 1833-23) (source KMI) 12 11 1 9 5 45 4 35 3 5 4 3 2 summer (May-October) sunshine precipitation in summer (May-October) ratio summer sunshine/summer precipitation 1991 1992 1993 1994 1995 1996 1997 1998 1999 2 21 22 23 24 source: Vogelbescherming Vlaanderen x source: KMI 16 14 12 1 8 6 4 2 16 14 12 1 8 6 4 2 16 14 12 1 8 6 4 2 Same requirements for proliferation Cyanobacteria
Problems in lakes of high recreational value Gavers, Harelbeke 3-4/27 Schulensmeer, Lummen 8-9/27
Microcystis ITS rdna diversity High local and regional ITS diversity of Microcystis blooms Average number of ITS genotypes per sample: 3.6 (range: 1-1). Correlation with environmental variables? Microcystis functional diversity Consequences for bloom dynamics and food web structure
Park Westveld, Sint-Amandsberg 25 2 15 1 5 % O2 ph 1, 9,5 9, 8,5 8, 7,5 7,
µg C L-1 Microcystis microscopic counts 25 2 solitary Microcystis cells Microcystis aeruginosa Microcystis viridis 15 1 5 15-5-7 29-5-7 12-6-7 26-6-7 1-7-7 24-7-7 7-8-7 21-8-7 4-9-7 18-9-7 2-1-7 16-1-7 3-1-7 M M
25 8 2 15 7 µg C L-1 Amoebae seem to cause a temporary bloom collapse and an abrupt change in population structure solitary Microcystis cells Microcystis aeruginosa Microcystis viridis amoebae 6 5 4 N ml -1 1 3 5 2 1 15-5-7 5-6-7 26-6-7 17-7-7 7-8-7 28-8-7 18-9-7 9-1-7 3-1-7
% % The amoebae seem to have certain food preferences are they selective feeders? 35 35 3 3 M. aeruginosa-type M. viridis-type amoebal infestation 'aeruginosa' amoebal infestation 'viridis' 1 1 9 9 8 8 25 25 7 7 N colonies ml -1 N colonies ml -1 2 2 15 15 1 1 5 5 6 6 5 5 4 4 3 3 2 2 1 1 1-5-7 15-5-7 29-5-7 12-6-7 26-6-7 1-7-7 24-7-7 7-8-7 21-8-7 4-9-7 18-9-7 2-1-7 16-1-7
Consequences for bloom toxicity? 25 2 µg C L-1 solitary Microcystis cells Microcystis aeruginosa Microcystis viridis total MC LR 3 25 15 2 15 µg l -1 1 1 5 5 15-5-7 5-6-7 26-6-7 17-7-7 7-8-7 28-8-7 18-9-7 9-1-7 3-1-7
A recurrent phenomenon? 25 2 Microcystis unicells M. aeruginosa-type Microcystis viridis-type amoebae 8 7 6 15 5 µg C L -1 4 N ml -1 1 3 5 2 1 1-5-8 22-5-8 12-6-8 3-7-8 24-7-8 14-8-8 4-9-8 25-9-8 16-1-8
% Influence of climatological conditions? N colonies ml -1 % 35 3 25 2 15 1 M. aeruginosa-type M. viridis-type amoebal infestation 'aeruginosa' amoebal infestation 'viridis' 1 9 8 7 6 5 4 3 5 2 1 1-5-8 15-5-8 29-5-8 12-6-8 26-6-8 1-7-8 24-7-8 7-8-8 21-8-8 4-9-8 18-9-8 2-1-8 16-1-8 3-1-8 35 1 3 9 8 N colonies ml -1 25 2 15 1 7 6 5 4 3 5 2 1 1-5-7 15-5-7 29-5-7 12-6-7 26-6-7 1-7-7 24-7-7 7-8-7 21-8-7 4-9-7 18-9-7 2-1-7 16-1-7
µg C L-1 Verification of specificity of amoebae grazing: experiments 25 2 Microcystis aeruginosa Microcystis viridis amoebae 8 7 6 15 1 5 15-5-7 15-7-7 15-9-7 15-11-7 15-1-8 15-3-8 15-5-8 15-7-8 15-9-8 5 4 3 2 1 N ml -1 Monocultures of different Microcystis-strains in WC-medium Amoebal cultures with mixed Microcystis-cultures as prey Cultivated at 24 C and 12/12 hour dark/light regime
First preliminary experiments 2 1 1 63. W168. W3. W32 auruginosa aeruginosa aeruginosa 1 W2 W2 W24 viridis viridis viridis 6 strains (3 M.viridis -type, 3 M. aeruginosa -type) 2 amoebal strains differences in biomass at beginning and end of incubation period measured by absorbance of chl a
Amoebal strain 1 p <,1 n.s. n.s.
Biomass (mg C/l) 8 Day 6 n.s. p =,9 p <,1 n.s. 7 6 5 4 3 without amoebae with amoebae 2 1 W168 W2 W2 W24 W3 W32 Microcystis-strains Amoebal strain 2
Conclusions and perspectives Grazing by amoebae can cause a temporary bloom collapse and a sudden shift in genetic structure of a Microcystis bloom. Preliminary experiments show food preference by the observed amoebae for different M. aeruginosagenotypes while M. viridis seems less susceptible. Future work includes the characterisation of the different amoebal strains and more standardized experiments to unravel genetic variation in Microcystis resistance against grazing by amoebae to complement patterns observed in nature.
Biomass M. aeruginosa (mg C l -1 ) Biomass M. viridis (mg C l -1 ) Voedselpreferentie-experimenten najaar 29 Invloed van Microcystis-stam en amoeben-stam (juli 29) 4 stammen M. aeruginosa: drie 1-cellig (W168, W3, W32), één kolonievormend (M31) 4 stammen M. viridis: allen kolonievormend (W2, W15, W2, W24) 4 amoeben stammen, twee dactylopoidale type, twee reticulate type 3 25 2 control +A1 +A2 +A16 +A21 3 25 2 control +A1 +A2 +A16 +A21 15 15 1 1 5 5 7.7.9 9.7.9 11.7.9 13.7.9 15.7.9 17.7.9 7.7.9 9.7.9 11.7.9 13.7.9 15.7.9 17.7.9 Sterk begrazingseffect op M. aeruginosa en niet op M. viridis
mg C l -1 9 8 7 6 5 4 3 2 1 W-168 amoeba 16 amoeba 1 amoeba 21 amoeba 2 control mg C l -1 14 12 1 8 6 4 2 W-15 amoeba 16 amoeba 1 amoeba 21 amoeba 2 control 12 6.7.9 9.7.9 13.7.9 17.7.9 7 6.7.9 9.7.9 13.7.9 17.7.9 mg C l -1 1 8 6 4 W-3 amoeba 16 amoeba 1 amoeba 21 amoeba 2 control mg C l -1 6 5 4 3 2 W-2 amoeba 16 amoeba 1 amoeba 21 amoeba 2 control 2 1 mg C l -1 18 16 14 12 1 8 6 4 2 6.7.9 9.7.9 13.7.9 17.7.9 W-32 amoeba 16 amoeba 1 amoeba 21 amoeba 2 control 6.7.9 9.7.9 13.7.9 17.7.9 mg C l -1 14 12 1 8 6 4 2 6.7.9 9.7.9 13.7.9 17.7.9 W-2 amoeba 16 amoeba 1 amoeba 21 amoeba 2 control 6.7.9 9.7.9 13.7.9 17.7.9 begrazingseffect niet homogeen voor M. aeruginosa, wel voor M. viridis
Biomass M. aeruginosa (mg C l -1 ) Biomass M. viridis (mg C l -1 ) Invloed van Microcystis-morfologie op amoeben begrazing (september 29) M. aeruginosa-stam M31: enkelcellig, kolonies, mengsel M. viridis-stam W24: enkelcellig, kolonies, mengsel Amoebenstam: A21 3 25 2 Colonies-A21 Colonies-Control Mixture-A21 Mixture-Control Single cells-a21 Single cells-control 3 25 2 Colonies-A21 Colonies-Control Mixture-A21 Mixture-Control Single cells-a21 Single cells-control 15 15 1 1 5 5 28.9.9 3.9.9 2.1.9 4.1.9 6.1.9 8.1.9 1.1.9 28.9.9 3.9.9 2.1.9 4.1.9 6.1.9 8.1.9 1.1.9 Morfologie speelt geen rol bij beide morphotypes
Graassnelheid van amoeben op M. aeruginosa (september 28) M. aeruginosa-stam M31: mengsel (15 mg C L-1) Amoebenstam: A21 (1. ind. ml-1) 12 dagen incubatie in donker 2 M31c-C 18 16 14 12 1 8 6 4 2 M31c-A Er vindt begrazing plaats, maar traag