J. Phycol. 50, 1009–1019 (2014) © 2014 Phycological Society of America DOI: 10.1111/jpy.12230 EUKARYOTIC PATHOGENS (CHYTRIDIOMYCOTA AND OOMYCOTA) INFECTING MARINE MICROPHYTOBENTHIC DIATOMS – A METHODOLOGICAL COMPARISON1 Bettina Scholz2,3,4 Institute of Chemistry and Biology of the Marine Environment, University of Oldenburg, Schleusenstrasse 1, Wilhelmshaven 26382, Germany Frithjof C. Kupper€ Oceanlab, University of Aberdeen, Main Street, Newburgh AB41 6AA, UK Wim Vyverman Department of Biology, Section of Protistology and Aquatic Ecology, University of Ghent, Krijgslaan 281 S8, Ghent 9000, Belgium and Ulf Karsten Institute of Biological Sciences, Applied Ecology & Phycology, University of Rostock, Albert-Einstein-Strasse 3, Rostock 18059, Germany Using sediment samples from the Solthorn€ tidal Abbreviations: AF, acid fuchsin; CR, Congo red; flat (southern North Sea, Germany), collected in CW, CalcoFluor White; FITC, fluorescein isothiocya- bi-weekly intervals from June to July 2012, a range nate; LCB, Lactophenol-cotton blue; MMS, Mayer’s of qualitative and quantitative screening methods Mucicarmine stain; MPB, microphytobenthos; NAG, for oomycete and chytrid pathogens infecting N-acetylglucosamine; NR, neutral red; PI, propidi- benthic diatoms were evaluated. Pre-treatment of um iodide; TB, Trypan Blue; WGA, wheat-germ agg- sediment samples using short ultrasound pulses and lutin gradient centrifugation, in combination with CalcoFluor White, showed the best results in the visualization of both pathogen groups. The highest Intertidal and shallow subtidal sediments are char- number of infected benthic diatoms was observed acterized by dense populations of benthic microal- in mid July (5.8% of the total benthic diatom gae, the so-called microphytobenthos (MPB; community). Most infections were caused by Paterson and Hagerthey 2001). These organisms chytrids and, in a few cases, oomycetes (Lagenisma play a key role in coastal ecosystem functioning, and Drebes (host: Coscinodiscus radiatus Ehrenberg) and contribute significantly to the primary production Ectrogella Zopf (hosts: Dimeregramma minor in in littoral zones (Pinckney and Zingmark 1993). Pritchard and Gyrosigma peisonis). Among the They are a major food source for the zoobenthos, chytrids, sporangium morphology indicated the and even for commercially important fish and shell- presence of five different morphotypes, infecting fish stocks as well as for migratory bird populations mainly epipelic taxa of the orders Naviculales (e.g., (e.g., Hillebrand et al. 2002). Most intertidal flats Navicula digitoradiata) and Achnanthales (e.g., are mainly colonized by diatom-dominated biofilms Achnanthes brevipes Agardh). The presence of (MacIntyre et al. 1996, Middelburg et al. 2000, Pat- multiple pathogens in several epipelic diatom taxa erson and Hagerthey 2001), which are usually com- suggests a significant role for fungal parasitism in posed of pennate forms, being either epipsammic affecting microphytobenthic diatom succession. or epipelic (e.g., Mitbavkar and Anil 2002). Besides abiotic parameters (e.g., Admiraal et al. Key index words: benthic diatoms; CalcoFluor White; 1984, Thornton et al. 2002), biotic factors such as chytrids; fluorescein isothiocyanate-labeled wheat-germ competition for light and nutrients and predator– agglutinin; oomycetes; sporangia; staining methods prey interactions have been shown to play key roles in the community structures of benthic diatoms (Thornton et al. 2002). In the marine environment, a growing body of evidence points additionally to 1 Received 11 May 2014. Accepted 31 July 2014. parasites as key players in the control of population 2 Present address: BioPol ehf., Einbuastig 2, Skagastrond€ 545, Iceland. dynamics and overall ecosystem structure (Gachon 3Present address: Faculty of Natural Resource Sciences, University et al. 2010). Several studies on planktonic microalgae of Akureyri, Borgir v. Nordurslod, Akureyri IS 600, Iceland. (e.g., Chambouvet et al. 2008) and benthic brown 4Author for correspondence: e-mail [email protected]. macroalgae (Kupper€ and Muller€ 1999, Kupper€ et al. Editorial Responsibility: P. Kroth (Associate Editor) 1009 1010 BETTINA SCHOLZ ET AL. 2006, Gachon et al. 2009, Strittmatter et al. 2013) collecting epipelic diatoms (e.g., De Jonge 1980), have shown the strong impact of eukaryotic patho- whereas for the sometimes abundant epipsammic gens such as oomycotes and chytridiomycotes on taxa, no standard method exists. In this study an their hosts’ ecology (e.g., Bruning 1991, Ibelings array of staining methods was compared which et al. 2004, Kagami et al. 2007, Gleason et al. 2008, allowed the identification of these pathogens using Sekimoto et al. 2008a,b). In particular, oomycetes different enrichment and staining procedures. Spe- infecting marine phytoplankton comprise several cifically, the detection of fungal pathogens in diluted marine representatives such as Lagenisma coscinodisci untreated surface sediment samples was compared Drebes, which was reported as an endobiotic parasite with those that had been pre-treated with ultrasonic of the centric diatom Coscinodiscus centralis Ehren- and gradient centrifugation. In addition, infection berg from the North Sea (Drebes 1966, 1968, Gotelli rates by eukaryotic parasites of the total microphyto- 1971). Furthermore, the endoparasitic, saprolegnia- benthic diatom communities and the impact on ceous oomycete Ectrogella Zopf is a parasite in dia- individual taxa were also calculated, depicting for toms and, according to Sparrow (1969), outbreaks of the first time qualitative and quantitative informa- Ectrogella perforans Petersen may attain epidemic pro- tion about the occurrence of such pathogens in the portions in the marine pennate diatom Licmophora marine benthic realm. Agardh. In contrast, chytrid infections seem to be most common among large species of freshwater MATERIALS AND METHODS phytoplankton that are fairly resistant to zooplankton grazing (Sommer 1987). Over 90% of all host cells in Study site and sample collection. The Solthorn€ tidal flat is a population may be infected, and every infection located in the eastern part of the Inner Jade, near the village ° 0 ″ ° 0 commonly leads to the death of the host cell (Canter of Tossens in Lower Saxony, Germany (53 34 2.03 N; 8 13 54.66″ E). One station was sampled at bi-weekly intervals dur- and Lund 1951, Ibelings et al. 2004, 2011). From brack- ing low tide from June 16th until July 14th 2012. Triplicate ish and marine ecosystems, only few representatives of surface samples of sediment were obtained by inserting Olpidium (Braun) Rabenhorst and Rhizophydium Schenk 8.5 cm diameter plastic petri dishes into the sediment to a have been described as parasites of small marine depth of 1 cm. All samples were stored at 4°C Æ 2°C until planktonic green algae and diatoms (Gleason et al. further processing in the laboratory. 2011). Chemicals. If not otherwise mentioned, all chemicals used The life cycles of oomycetes and chytrids begin in this study were of the highest purity from Sigma/Aldrich Chemical Co. (Sigma-Aldrich Laborchemikalien GmbH, with the attachment of a motile zoospore to the Seelze, Germany). surface of an algal host cell. Besides differences in Treatment of sediment samples. The sediment samples were zoospore morphology, there are fundamental differ- prepared in four subsequent steps (Fig. 1). In a first step, ences between both groups of parasites. Chytrids are three sediment subsamples (volume: 1 cm3) were each diluted in 40 mL sterilized artificial seawater, using Tropic true fungi, while oomycetes are stramenopiles, a het- â erotrophic sister group, for example, brown algae Marin (GmbH Aquarientechnik, Wartenberg, Germany), and diatoms (Gleason et al. 2011). In consequence, with a salinity of 30 and a pH of 8.0, respectively (Fig. 1A). To remove epipsammic diatom taxa from the sediment parti- for example, chytrids are characterized by chitina- cles, ultrasonic pulses of 3 9 2 s were used in a second step ceous cell walls, whereas oomycetes have predomi- (ultrasonic processor UP50 Dr. Hielscher GmbH, Tetlow, nantly cellulosic walls (consisting mainly of 1,3-b- Germany, amplitude of 40% at 0.5 s intervals, Fig. 1B). Subse- glucans, some 1,6-b-glucans and 1,4-b-glucans). Chi- quently, the mixtures were separated in a third step with a tin, which is a major constituent of fungal cell walls, slightly modified version of the technique described by De has been detected in small amounts in only a few Jonge (1979) (Fig. 1C). The procedure followed is based on oomycetes (Latijnhouwers et al. 2003). Due their density gradient centrifugation of the samples in Ludox-TM. Benthic diatom species were harvested from the 70% Ludox- small and holocarpic thalli, microscopic identifica- TM layer, washed twice with 2.5 mL of the seawater solution tion of these parasitic species is not easy and often by centrifugation (10 min at 500 rpm, Centrifuge EBA 20, requires ultrastructural characterization of zoospores Hettich GmbH, Tuttlingen, Germany) and placed in glass for absolute certainly (Barr 1981, Longcore 1995, Utermohl€ counting chambers in a fourth step (100 lL, Hanic et al. 2009, Letcher and Powell 2012). Fig. 1D). Microscopic observation of the residues (sediment Detection of eukaryotic pathogens infecting phyto- particles) using epifluorescence confirmed the almost com- plete release of the cells (Olympus BX51, equipped with a plankton species can be difficult, and different stain- BX-RFA reflected fluorescence system,