Eukaryotic Pathogens (Chytridiomycota and Oomycota) Infecting Marine Microphytobenthic Diatoms

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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 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, Hamburg, Germany). ing methods exist. Among these CalcoFluor White 800 lL(89 100 lL) of the samples from steps one and (CW) and wheat-germ agglutin (WGA) conjugated two (after sonification) were used for a first screening with fluorescein isothiocyanate (FITC) followed by using autofluorescence and the staining methods described observation unan epifluorescence microscope are below. frequently used for the visualization of fungal para- Staining methods. Both parasites attached to diatom cells sites (e.g., Muller€ and Sengbusch 1983, Rasconi (epibiotic) as well as endobiotic ones were stained according to different methods and protocols (see Table 1). In total ten et al. 2009, Marano et al. 2012). For benthic sam- staining methods were tested at the beginning of this study, ples, there is the additional challenge to separate using samples from each step of the sediment preparation intact cells from inorganic and organic particles. (cf. Fig. 1) of which only the FITC-WGA and CW protocols Here, the lens-tissue method is routinely used for were modified. For the use of FITC-WGA, 3 mL of borate EUKARYOTIC PATHOGENS INFECTING MPB DIATOMS 1011

FIG. 1. Illustration of the methods used for the collection of surface sediment samples in the Solthorn€ tidal ﬂat (A) as well as the sub- sampling scheme for each step of the preparation (B–D). * vials were prepared according to Schrader (1974).

buffer (10 mM; pH 7.4) were added to the samples. For the (1884), Sparrow (1960), Johnson and Sparrow (1961), Drebes preparation of a 1.0 mg mL 1 stock solution, 5.0 mg of (1966), Karling (1977) and Letcher and Powell (2012) were lyophilized FITC-WGA in 5.0 mL of phosphate-buffered sal- used. For diatom species determination, samples of the slurry ine (PBS) were dissolved and stored at 18°C (stable for (3 9 5 mL) were prepared according to the method of Sch- 1 month). For the assay, the stock solution was diluted in rader (1974) and embedded in Naphrax (refractive index PBS to a final concentration of 7.0 lg mL 1. 1 mL of this 1.74, Northern Biological Supplies Ltd., Ipswich, UK). For solution was used for labeling. After incubation (30 min; species identification the following literature was used: Cleve 25°C) on a rotary shaker (130 rpm), samples were rinsed (1894, 1895), Hasle and Syvertsen (1996), Hendey (1964), twice with fresh buffer, diluted to a final volume of 5 mL and Hustedt (1927–1966, 1939), Krammer and Lange-Bertalot examined at 4009 magnification. In contrast, 150 lL of 10% (1986–91), Kutzing€ (1844, 1849), Schmidt et al. (1874–1959), KOH solution and 150 lL of 0.2% CW were added to 1 mL Witkowski (1994) and Witkowski et al. (2000). All permanent samples in Utermohl€ glass counting chambers and diluted to samples were examined una light microscope (Axiophot, Carl a final volume of 5 mL, after which the samples were incu- Zeiss AG, Oberkochen, Germany) at 1,0009 magnification. bated for 10 min at room temperature. All assays were con- Numerical processing of the identified diatom species was ducted in glass Utermohl€ counting chambers, because some carried out after classification (classes and orders only) of the staining agents (e.g., WGA) were observed to bind to according to the taxonomic nomenclature described in plastic material. The samples were counted at 4009 magnifi- Medlin and Kaczmarska (2004). cation unan inverted fluorescence microscope with UV excita- Calculation of the infection rates. The percentage of infected tion and also with visible light (Olympus IX53). Diatom cells was calculated by dividing the number of infected cells species were identified as far as possible, enumerated and by the total number of host cells. The mean number of chytr- assigned to taxonomic entities. Abundance was calculated as ids and oomycetes per cell (host) in the diatom population the number per cm2 sediment surface. The results of the cell was also calculated, by dividing the total number of parasites counts for the June and July samples (June 6, July 1 and 14, attached to algal cells by the total number of host cells, to 2012), comprising all staining methods used, are given in Fig- normalize the cell density among treatments. This value is ure 2. referred to as the mean intensity of infection (Holfeld 2000), Identification of eukaryotic pathogens and their hosts. Sub-sam- reflecting the number of pathogens that succeed in attaching ples (3 9 50 lL) from all staining preparations were to their host. mounted in Slowfade-Light antifade solution (MoBiTec, Statistical analysis. Percentage infection and infection Gottingen,€ Germany), whereas in parallel aliquots intensity of eukaryotic parasites obtained from each treat- (3 9 500 lL and 10 lL, respectively) of the unstained sediment (untreated sediment samples as well as ultrasound-trea- ment samples after sonification and the enrichment step were ted and gradient-centrifuged ones) were compared using dried and embeded in Euparal (Chroma Gesellschaft, Schmid one-way analysis of variance (ANOVA) followed by Tukey’s GmbH, Kongen,€ Germany; Fig. 1). For identification of tests. The data were checked for normality with Bartlett’s test eukaryotic parasites the studies of Schenk (1858), Zopf (Bartlett 1947) and, where necessary, log transformed prior 1012

TABLE 1. Overview of the staining methods used during the surveys and their effectiveness in the detection of oomycetes and chytrids in the samples.

Abbre- Reference including Staining agent viation Supplier staining protocol Staining specificity Visibility of pathogen structures in this study Acid fuchsin AF F8129; Dickson et al. 2003 Stains keratin, cytoplasm, and Nuclei from active cells, but no empty sporangia. Sigma-Aldrich nuclei Too time consuming, due to lack of specificity CalcoFluor White CW 18909; Herth 1980 Binds to cellulose and chitin Sporangia and empty sporangia visible. Also Sigma-Aldrich in cell walls structures of oomycetes were clear visible. High back staining in sediment samples Congo red CR C6767; Briggs and Binds to chitin in cell walls Sporangia and empty sporangia visible. Sigma-Aldrich Burgin 2004 No oomycetes detected. High back staining in AL. ET SCHOLZ BETTINA sediment samples FITC-conjugated wheat WGA FL-1021; Biozol Harris 2005 Chitin-specific binding protein Sporangia and empty sporangia visible. germ agglutinin No oomycetes detected. High back staining in sediment samples Lactophenol-cotton LCB PL.7054; Pro-Lab Leck 1999 Cotton blue stains the chitin in Sporangia and empty sporangia visible. blue Diagnostics the fungal cell walls No oomycetes detected. High back staining in sediment samples Lactophenol-Picric LPA 41506; Fleming and Picric acid binds to No chytrids and oomycetes were detected. Acid Solution Sigma-Aldrich Smith 1944 chitin-chitosan complexes High back staining in sediment samples Mayer’s Mucicarmine MMS 41325; Walsh and Jass 2000 Stain for carbohydrates Not specific, stained all organisms containing Stain Solution Sigma-Aldrich carbohydrates. High back staining in sediment samples Neutral red NR N2889; Vierheilig et al. 2005 Stains lysosomes No empty sporangia visible. Too time consuming, Sigma-Aldrich due to lack of specificity Propidium iodide PI P4864; Riccardi and DNA stain; used as a nuclear No empty sporangia visible. Too time consuming, Sigma-Aldrich Nicoletti 2006 counter stain due to lack of specificity Trypan Blue TB T8154; Phillips and Binds to chitin in cell walls Sporangia and empty sporangia visible. Sigma-Aldrich Hayman 1970 No oomycetes detected. High back staining in sediment samples EUKARYOTIC PATHOGENS INFECTING MPB DIATOMS 1013

(probably due to the presence of chitin in benthic detritus). According to the manufacturer’s descrip- tion CW is a non-specific fluorochrome that binds to cellulose and chitin in cell walls, whereas WGA is a sugar-binding protein (lectin) that binds only to a series of consecutive N-acetylglucosamine residues (Roth 1978). In general, chitin rivals cellulose as the most abundant biopolymer in nature (Meng et al. 2012) and planktonic crustaceans have been considered the most significant source of chitin in the marine environment (Souza et al. 2011). In addition, copepod fecal pellets are encased in chitin (Yoshikoshi and Ko 1988) and several common phytoplankton genera of diatoms, such as Thalassiosira Cleve and Skeletonema Greville, produce chitin as a significant portion of their biomass (up to 33%, Smucker and Dawson 1986). According to Boyer (1994) much of the chitin found in oceans is rapidly degraded while in suspension, but some is also incorporated into sediments (up to 20%, depending on the sediment composition). Thus it is highly likely that the high backscattering effects observed in this study during the use of untreated FIG. 2. Comparison of autofluorescence counting and the ten surface sediment samples in combination with chi- staining methods used during three sampling events for untreated as well as pre-treated surface sediment samples from tin-specific staining agents were caused by accumu- the Solthorn€ tidal flat, presenting the prevalence (= ratio of lated chitin residues originating from the infected cells). The individual staining methods are given in planktonic realm. Table 1. Considered were the main epi- and endobiotic eukary- The further pre-treatment of the sediment sam- otic parasite structures such as thalli, sporangia, and rhizoidal sys- ples using ultrasound and gradient centrifugation tems. Mean values ( SD), based on triplicate assays and the samplings of one station from June to July 2012, are given. resulted in an increase in the visualization of parasitic structures (rhizoidal systems, thalli and sporangia), due to elimination of the backscattering to analysis. A P-value of <0.05 was considered as significant. effects. Both methods, ultrasound for the mechani- All tests were performed with the program SPSS Statistical cal disruption of diatom-sand agglomerates as well SoftwareTM, version 11.5 (Edinburgh, UK). as density gradient centrifugation for the separation of sediment grains from the benthic diatom biomass, have already been applied by several scientists RESULTS AND DISCUSSION to gain information about the community composi- Sediment sample pre-treatment and staining tion of epipelic and epipsammic diatom taxa inhab- methods. Initially, all ten staining methods used in iting intertidal sediment surfaces (e.g., Admiraal combination with the untreated sediment samples 1984, Mel eder et al. 2005, Scholz 2014). In contrast were more or less ineffective, being not specific to these protocols, only short ultrasound pulses enough and/or giving high backscattering effects (3 9 2 s) were used in this study, to conserve the with the sediment matrix (recovery rates only up to fine structures of the eukaryotic parasites (thalli and 3.9%, Fig. 2; Table 1). Samples stained with Mayer’s attached sporangia), which were found to be Mucicarmine stain (MMS) showed positive color deformed or destroyed by pulses longer than 10 s reactions with the exopolysaccharides (polymeric (data not shown). carbohydrates) originating from the benthic dia- Generally, the highest numbers of epi- and endo- toms and the sediment matrix, whereas propidium biotic parasites infecting benthic diatoms were iodide (PI), neutral red (NR) or acid fuchsin (AF) recorded by the use of CW (up to 46% and 68% stained only all living organisms which were present higher in comparison to the untreated sediment = in the unpreserved sediment samples (e.g., algae, samples, ANOVA: F1,90 32.9, P<0.0001, Fig. 2), bacteria, cilliates, heterotrophic nanoflagellates). whereas the stains FITC-WGA, CR, LCB and TB Conversely, empty sporangia and inactive cells, such showed significantly lower recovery rates (up to 43% as those after an infection, were not stained by these lower than the CW stained samples, ANOVA: = methods. In particular, staining agents specific for F1,15 39.8, P<0.0001). In addition, the high num- chitin and its polymers such as CW, Congo red bers of presumed infections using the CW stain can (CR), Lactophenol-cotton blue (LCB), Trypan Blue be explained by the counting of both groups: chytr- (TB) and WGA produced a manifold of false posi- ids and oomycetes, whereas FITC-WGA, CR, LCB tive signals with the fine and muddy sediments and TB seemed to be only sensitive for the chitin 1014 BETTINA SCHOLZ ET AL.

TABLE 2. Total diatom abundances (classes and orders according to the taxonomic nomenclature described in Medlin and Kaczmarska 2004) and number of epibiotic and endobiotic eukaryotic pathogens. Data were obtained from overview countings in Utermohl€ chambers, using CalcoFluor White as staining agent. Mean values ( SD) for all subsamples are given (n = 9).

Diatoms/Sampling date June 16, 2012 July 01, 2012 July 14, 2012 Magnitude Class: Coscinodiscophyceae 0.14 0.05 0.12 0.1 0.19 0.05 [103 cells cm 2] Class: Mediophyceae 0.8 0.1 0.3 0.1 1.0 0.4 [103 cells cm 2] Class: Bacillariophyceae Order: Achnanthales 2.3 1.1 2.4 1.2 2.1 1.5 [103 cells cm 2] Order: Bacillariales 1.3 1.0 0.8 0.4 1.0 0.6 [103 cells cm 2] Order: Naviculales 3.6 1.1 3.1 0.8 2.8 0.9 [104 cells cm 2] Order: Rhaphoneidales 0.3 0.01 0.23 0.01 0.42 0.02 [102 cells cm 2] Order: Striatellales 0.42 0.02 0.38 0.01 0.59 0.02 [102 cells cm 2] Order: Thalassiophysales 1.4 0.4 1.2 0.5 1.1 0.6 [103 cells cm 2] ∑ 4.21 3.8 3.71 3.1 3.35 4.1 [104 cells cm 2] Pathogens Epibiotic 9.2 0.2 12.1 0.1 19.4 0.6 [102 cells cm 2] Endobiotic 3.8 0.2 4.2 0.3 2.2 0.3 [cells cm 2] ∑ 9.23 0.4 12.15 0.4 19.47 0.9 [102 cells cm 2] Infection rates 2.2% 3.4% 5.8%

TABLE 3. Morphological characteristics of the epi- and endobiotic eukaryotic pathogens obtained from analysis of permanent slides, using sediment samples collected in the Solthorn€ tidal ﬂat in June and July 2012.

Pathogen Morphological characteristics Suggestions Chytridiomycota (monocentric, holocarpic or eucarpic, epibiotic) Type I Sporangium: sessile, ovoid or spherical (globose), 14–25 lm Rhizophydium Schenk (1858), Sparrow (1960), high, 14–26 lm diameter; wall thin, and smooth, colorless, section 1 in Letcher and Powell (2012); double-contoured, 1–1.5 lm thick, with a broad apical or sub similarities with R. brevipes var. marinum apical papilla. Rhizoid: simple, short, thick, and peg-like, (but sporangia seems to be too small); resting projecting only slightly beyond the inner part of the host wall spores not observed Type II Sporangium: sessile, ovoid-obpyriform or urceolate (empty Rhizophydium Schenk (1858), Sparrow (1960), sporangia), 5–11 lm high by 5–9 lm in diameter; thick walled section 2 in Letcher and Powell (2012); resting (2.5–3 lm), wall smooth, colorless; Rhizoid: rhizoids spores not observed not observed Type III Sporangium on a short thick walled, rigid stalk Chytridium sp. Johnson and Sparrow (1961), (extrametrical, 1.3–2.5 lm long), ovate or ellipsoid, 12–15 lm according to Sparrow (1960) similarities with long by 9–11 lm high by 8–9 lm wide; without rhizoids, C. chlorobotrytis Fott with exception of stalk; usually with a spherical “oil” drop like spot (3–4 lm), resting spores not observed gregarious Type IV Sporangium on a thin walled, flexible stalk (extrametrical, Chytridium sp. Sparrow (1960), Johnson and 5–12.5 lm long), ovoid, obpyriform or spherical, 9.5–15.5 lm Sparrow (1961); resting spores not observed long by 9–15.5 lm in diameter, with a thin, smooth, colorless wall; gregarious, rhizoidal system short, delicate, and sparsely branched Type V Sporangium on a flexible stalk (extrametrical, 12–44 lm Chytridium or Rhizophydium (Sparrow 1960), long), globose, 20–35 lm in diameter, hyaline, smooth, in general both groups include species with somewhat thick-walled (2–3 lm thick) solitary; rhizoid simple, stalks (Sparrow 1960, Johnson and Sparrow short, thick, and peglike, projecting only slightly beyond the 1961, Letcher and Powell 2012); resting inner face of the host wall spores not observed Oomycota (monocentric, holocarpic, endobiotic) Type I Sporangium single-celled, non-branched, inoperculate, Ectrogella Zopf (1884) or Olpiopsis sp. (!), because spherical, lenticular 26–40 lm long by 20–35 lm in diameter, both are known to form single-celled, with a thin colorless wall, discharge tubes from one to five, non-branched endiobiotic sporangia in the host broadly conical, 8–10 lm long by 9–12 lm in diameter cytoplasm (Sparrow 1960), the two genera differ only in spore morphology Type II Thallus tubular, very thin-walled, 170–350 lm long by Lagenisma Drebes (1966), according to Sparrow 5–7.5 lm in diameter, infection tube occasionally persistent (1960) similarities to Lagenidium sp. containing cell walls of chytrids. Conversely, the dif- pre-treated sediment samples, suggesting the lack of ferences in the susceptibility of the staining agents chitin in the oomycetes found during the present used in this study in conjunction with the composi- short-term monitoring. tion of chytrid and oomycete cell walls (chitin vs. Many actinomycetes and fungi are known to fluo- cellulose) could be the crucial factor for the non resce in UV (e.g., Rost 1995). Particularly under detection of oomycetes in the untreated as well as violet excitation, weak to moderate fluorescence in EUKARYOTIC PATHOGENS INFECTING MPB DIATOMS 1015

FIG. 3. Examples of diatoms infected by different chytrids found in the Solthorn€ tidal flat. Samples were prepared as described in the methods & material part, using the modified CW method. Black bars indicate 10 lm. Pictures A, B, E and F were taken under fluorescence light, whereas for C and D usual visible light was used. (A) Cylindrotheca closterium/chytrid type II; (B) Amphora exigua/chytrid type IV; (C) Navicula digitoradiata/chytrid type III; (D) Achnanthes brevipes/ chytrid type V; (E) Navicula gregaria/ chytrid type I; (F) Diploneis didyma/ chytrid type II.

fruiting bodies has been observed in several fungal Liebezeit 2012) it was also tested in connection to species, suggesting that autofluorescence is a com- the screening for eukaryotic parasites in this study. mon feature of these taxa (Rost 1995). While for Here, autofluorescence yielded only poor results in some species fluorescent substances such as orella- all cases, which might be caused by the general lack nine (e.g., Cortinarius orellanus, Keller-Dilitz et al. of fluorescing structures and/or the damage and 1985) or leprocybin (subgenus Leprocybe, Kopanski denaturation of chloroplasts and fluorescing sub- et al. 1982) are described, no references about such stances, respectively, during the pre-treatments. substances were found for chytrids and oomycetes. A clear disadvantage of the pre-treatments was the However, due to the fact that autofluorescence was loss of free swimming zoospores during the prepara- used in previous investigations regarding the MPB tion steps, which are in most cases necessary for the composition in the Solthorn€ tidal flat (Scholz and species identification of chytrids and oomycetes 1016 BETTINA SCHOLZ ET AL.

the second most abundant order in the benthic diatom community (6.1%), whereas the Thalassiophy- sales (3.3%) and Bacillariales (2.8%) represented the third and fourth most abundant group of taxa, respectively. All other groups had significantly lower total abundances and can be regarded as minor groups of the MPB diatom communities of the Sol- thorn€ tidal flat during this short sampling period. Only few diatoms were found to be infected by eukaryotic pathogens, with the highest prevalence recorded in mid July 2012 with 5.8% of the overall benthic diatom community (Table 2). At all three dates of the survey, the numbers of epibiotic parasites exceeded the occurrence of endobiotic ones by far, ranging from 99.6% to 99.8% in June and July 2012, respectively. Generally, chytrids are frequently found to cause significant algal mortality in freshwater plankton, where during epidemics more than FIG. 4. Examples of diatoms infected by different oomycetes 90% of the host population may be infected (e.g., € found in the Solthorn tidal flat. (A) Lagenisma Drebes (host: Cos- Van Donk and Ringelberg 1983, Kagami et al. cinodiscus radiatus Ehrenberg) and (B) Ectrogella Zopf (host: Gyr- osigma peisonis [Grunow] Hustedt). Black bars indicate in (A) 30 2006). Regarding the impact of oomycetes on their lm and (B) 15 lm. host population, it was reported for instance that an approx. 13% infection prevalence in a natural popu- (Sparrow 1960). An important aspect of the present lation of Coscinodiscus was caused by L. coscinodisci approach is the use of settling chambers for detect- Drebes in the Weser estuary of northern Germany ing eukaryotic pathogens according to the classical (Raghukumar 1996). method of Utermohl€ (1958). Here, the identification Epi- and endobiotic infections of benthic diatoms. Due of species was in most cases impossible. Similar to the methods used, identification of epi- and endo- results were obtained by Rasconi et al. (2009), who biotic eukaryotic pathogens was only possible at found that the main reason for this is generally that genus level. Generally, the species identification of staining directly in the Utermohl€ chamber resulted eukaryotic parasites in environmental samples is very in very poor quality of parasite visualization. How- difficult as they rarely show species-specific ever, the use of settling chambers in this study was morphological structures (Miller 1968) and observa- reduced to overview counts, distinguishing only tions in samples obtained from a habitat generally do between epi- and endobiotic parasites (Table 2), not allow observation of all phases of their full life while for identification purposes permanent slides cycle. Most of these pathogens have not been estab- were prepared (Table 3; Fig. 3). In this context, the lished in pure culture yet where they could be studied use of polycarbonate filters (0.6 lm pore-size), which extensively under controlled and variable conditions are shown by Rasconi et al. (2009) to be practical for to reveal life cycle transitions. In addition, differences freshwater phytoplankton samples, may be also use- between the morphology of in situ observations and ful in future investigations of MPB samples after a species in culture have been found for some repre- pre-treatment such as applied in this study. sentatives of the Chytridiomycota (e.g., Chen and Total benthic diatom community and infection rates. Chien 1996), which can be explained, for example, The total diatom abundance did not vary signifi- by nutrient effects (e.g., Hasija and Miller 1971, Chen cantly during the short-term monitoring from June and Chien 1996). These environmental influences on to July 2012 (P > 0.05), decreasing only slightly over morphology further complicate the identification of the three sampling events (difference from 16 June parasitic species in environmental samples. = = to 14 July, 2012 7.6%, ANOVA: F1,16 34.7, Overall five representatives of the Chytridiomycota P<0.0001, Table 2). The data obtained from the were distinguished according to their rhizoidal sys- overview countings, using Utermohl€ chambers in tem and epibiotic sporangia (Table 2; Fig. 3). The combination with the CW stain, revealed the Navi- diatom taxa infected by chytrids comprised repre- culales as the most abundant group within the ben- sentatives of the Bacillariales, Naviculales, Achnant- thic diatom community of the Solthorn€ tidal flat hales and Thalassiophysales (Fig. 3). Specifically, (up to 85.3% of the total diatom abundances). Gen- Navicula digitoradiata (Gregory) Ralfs (Fig. 3C) was erally, the dominance of small species belonging to infected by chytrid type III, whereas two other the genus Navicula spp. appears to be an important species of this order were found to be infected by feature of European intertidal MPB (e.g., Admiraal chytrid type I (N. gregaria Donkin, Fig. 3E) and II et al. 1984, Underwood 1994, Hamels et al. 1998). (Diploneis didyma Ehrenberg, Fig. 3F). Additionally, Besides the dominance of the representatives of chytrid type II was also observed on cells of Cylindrot- the Naviculales, the Achnanthales were identified as heca closterium (Ehrenberg) Reiman & Lewin EUKARYOTIC PATHOGENS INFECTING MPB DIATOMS 1017

(Fig. 3A) a representative of the Bacillariales. Fur- Canter, H. M. & Lund, J. W. G. 1951. Studies on plankton para- thermore, one representative of the Achnanthales sites. III. Examples of the interaction between parasitism and other factors determining the growth of diatoms. Ann. Bot. (Achnanthes brevipes Agardh, Fig. 3D) was infected by 15:359–71. chytrid type V, while one representative of the Tha- Chambouvet, A., Morin, P., Marie, D. & Guillou, L. 2008. Control lassiophysales (Amphora exigua Gregory, Fig. 3B) was of toxic marine dinoflagellate blooms by serial parasitic kill- infected by type IV. It has to be pointed out, how- ers. Science 322:1254–7. ever, that the taxonomic identity of these taxa have Chen, S. F. & Chien, C. Y. 1996. Morphology and zoospore ultrastructure of Rhizophydium macroporosum (Chytridiales). Taiwa- to be revised (especially when molecular methods nia 41:105–12. will become available) considering recent studies on Cleve, P. T. 1894. Synopsis of the naviculoid diatoms. Part 1. what are likely closely related pathogens in brown Kongliga Svenska Vetenskaps-Akademiens Handlingar 26:1–184. algae (Kupper€ et al. 2006). Cleve, P. T. 1895. Synopsis of the naviculoid diatoms. Part 2. Kongliga Svenska Vetenskaps-Akademiens Handlingar 27:1–220. In addition, microscopic analysis of the endobiotic De Jonge, V. N. 1979. Quantitative separation of benthic diatoms thalli and sporangia, using permanent slides, from sediments using density gradient centrifugation in the revealed two representatives of the Oomycota (cf. colloidal silica Ludox-TM. Mar. Biol. 51:267–78. Table 2, Fig. 4), Lagenisma Drebes (Lagenismatales, De Jonge, V. N. 1980. Fluctuations in the organic carbon to chlo- host: Coscinodiscus radiatus Ehrenberg, Coscinodisco- rophyll a ratios for estuarine benthic diatom population. Mar. Ecol. Prog. Ser. 2:345–53. phyceae, Fig. 4A) and Ectrogella Zopf (Saprolegniales, Dickson, S., Schweiger, P., Smith, S. 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