Mycologia, 99(2), 2007, pp. 215–221. # 2007 by The Mycological Society of America, Lawrence, KS 66044-8897 Molecular identification and classification of Cochlonema euryblastum, a zoopagalean parasite of Thecamoeba quadrilineata Martina Koehsler Key words: 18S rDNA, endocytobiont, free-living amoeba, parasite, Zoopagales, Zygomycota Department of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology, Medical University of Vienna, Kinderspitalgasse 15, 1095 Vienna, Austria INTRODUCTION Free-living amoebae (FLA) of the genus Thecamoeba are often called ‘‘amoebae with a pellicle’’ because of their thick fibrous or amorphous glycocalyx (Penard 1905). Based on their locomotive morphology they are divided into smooth, rough and wrinkled groups. Species of Thecamoeba are distributed widely in freshwater, can occur in saltwater and are among the largest protozoa in soil and mosses. Apparently no cysts are formed, despite their occurrence in habitats subject to drying (Page 1977, 1991). FLA are known to serve a great variety of pathogenic and nonpathogenic bacteria as vehicles or hosts. In the past years particularly Acanthamoeba spp. harboring bacteria, such as Legionella pneumophila, Mycobacterium spp. or Chlamydia-like bacteria and protecting them against unfavourable environmental conditions, have been in the center of interest (Greub and Raoult 2004). However, other FLA are suitable hosts, such as Hartmannella vermiformis, Balamuthia mandrillaris and Naegleria spp. (Donlan et al 2005; Hoffmann and Michel 2001; Horn et al 2000; Michel et 1999, 2005; Shadrach et al 2005; Walochnik et al 2005). Interactions of FLA with fungi have been described less frequently, nevertheless fungi are known to parasitize in FLA, usually killing their host. In 1879 Leidy described an amoeba with appendices resembling strings of sausages, which in fact belonged to the genus Mayorella, parasitized by Amoebophilus simplex (Brief 2005). Charles Drechsler described various Phytomyceta and Zoopagaceae parasitizing and killing terricolous amoebae (Drechsler 1938, 1942). Furthermore he identified a higher fungus belonging to the Basidiomycota as a parasite of Amoeba terricola (Drechsler 1969). More recently, a natural infection of Vannella spp. with a microsporidian species, growing and multiplying in its host, has been reported (Hoffman et al 1998). In an interaction of Acanthamoeba spp. with Cryptococcus neoformans the amoeba was shown to represent not only a nourishing host but to prime the fungus for an intracellular pathogenic strategy in macrophages (Steenbergen et al 2001). In 1998 Michel reported on a yeast-like endocytobiont in Thecamoeba similis and in 1999 he described Rolf Michel Central Institute of the Federal Armed Forces Medical Services, Department of Parasitology, Andernacher Straße 100, 56070 Koblenz, Germany Claudia Wylezich Department of Ecology and Limnology, Institute of Zoology, University of Cologne, Weyertal 119, 50923 Cologne, Germany Johannes Lugauer Central Institute of the Federal Armed Forces Medical Services, Department of Parasitology, Andernacher Straße 100, 56070 Koblenz, Germany Julia Walochnik1 Department of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology, Medical University of Vienna, Kinderspitalgasse 15, 1095 Vienna, Austria Abstract: Free-living amoebae can serve a great variety of organisms, predominantly bacteria but to a certain extent also fungi, as a suitable host supplying them with nutrients and protecting them from adverse environmental conditions. In the current study 18S rDNA sequencing was performed to identify a fungal parasite in a Thecamoeba quadrilineata isolate. This parasite morphologically resembled Cochlonema euryblastum, a member of the order Zoopagales, which comprises parasitic species on fungi and invertebrates. Sequence analysis corroborated the morphological identification and the fungal parasite clearly can be assigned to the Zoopagales. Phylogenetic analysis revealed C. euryblastum clustering with two representatives of the mycoparasitic family Piptocephalidaceae. This zooparasitic-mycoparasitic clade represents a sister group of a clade including another member of the Piptocephalidaceae and two other zooparasitic families. Thus, the addition of C. euryblastum to the zoopagalean tree further confirms the finding that molecular data do not support the traditional classification of the Zoopageles. Accepted for publication 14 Nov 2006. 1 Corresponding author. E-mail: Julia.walochnic@meduniwien.ac.at 215 216 MYCOLOGIA a fungus-like endocytobiont in a Thecamoeba quadrilineata isolate, which originally had been isolated from an eaves gutter. This endocytobiont appeared as a coil-shaped structure immobilizing and killing the amoebae. This endocytobiont morphologically resembled Cochlonema euryblastum, a parasitic fungus destructive to soil amoebae described by Drechsler in 1942 (Michel and Wylezich 2005). The aim of the current study was to proof the morphological identification of this fungal parasite in T. quadrilineata by employing 18S rDNA sequencing and to reveal the position of this fungus within the order Zoopagales. To date no sequence data of the whole family Cochlonemataceae are available. Consequently 18S rDNA sequences of several other zoopagales were used for the construction of a phylogenetic tree. MATERIAL AND METHODS Amoebae.—The Thecamoeba quadrilineata strain was isolated in 1999 out of a sediment sample from an eaves gutter in Neuwied/Rhein in Germany. The original isolate contained a conspicuous parasitic fungus, which was identified as Cochlonema euryblastum based on morphologic characters. Culture of amoebae/fungi.—Amoebae were cultivated on nonnutrient agar plates, containing 0.1% sea salt and covered with a lawn of Escherichia coli. New subcultures of amoebae were prepared on a weekly basis. Because the fungal infection was lethal to the amoebae after a certain period of time, a parallel culture without fungus was installed to maintain the amoebal strain. This was achieved by adding an antimycotics suspension to the plate cultures until the amoebae were free of fungi. In addition, to maintain the fungus noninfected amoebae cultures were reinfected with the fungus when necessary. Re-infection was achieved by excising a piece of agar from a plate covered with conidia and carefully sliding the agar piece over amoebic cultures. Approximately 4 d after infection the majority of amoebae were dead and a large number of coilshaped fungi surrounded by single conidia were observable. Amoebae were considered dead when the cell membrane was disrupted. About 1 wk after infection amoebae/ fungi were harvested for DNA isolation. Electron microscopy.—Amoebal host cells were harvested 3 d postinfection. The cell suspension was centrifuged at 6003 g for 10 min. The resulting pellet was fixed in 3% glutaraldehyde (1 h), transferred to 0.1 M cacodylate buffer, postfixed in 1% osmium tetroxide and embedded in Spurr resin. Sections were stained with uranyl acetate and Reynold’s lead citrate and examined with a Leo EM 910 electron microscope. Isolation of DNA.—Uninfected and infected amoebae were harvested directly from the plate cultures with sterile cottontipped applicators and resuspended in 200 mL sterile distilled water. Cells were disrupted by multiple freeze- thawing in liquid nitrogen before whole-cell DNA was isolated by a modified UNSET procedure (Hugo et al 1992). In brief, 500 mL of UNSET lysis buffer was added to the amoebae and fungi suspended in 200 mL distilled water. Then the suspension was overlaid with 700 mL of phenolchloroform-isoamyl-alcohol (PCI) and shaken gently overnight. The suspension was centrifuged at 30003 g for 10 min, and the upper, aqueous phase was transferred to a new tube. PCI extraction was repeated two times for 10 min each time. Nucleic acids were precipitated by ethanol (overnight at 220 C), pelleted at 12 0003 g for 30 min at 4 C, washed in 70% ethanol, air dried, and resuspended in 30 mL of sterile distilled water. DNA Amplification.—For DNA amplification of the 18S rRNA gene, primers SSU1 (59-CGACTGGTTGATCCTGCCAGTAG-39) and SSU2 (59-GTGAACCTGCAGAAGGATCAGGA-39), complementary to the 59- and the 39- end of the gene, respectively (Gast et al 1996), and six internal primers (Walochnik et al 2004), P1fw (59-CAAGTCTG GTGCCAGCAGC-39), P1rev (59-GCTGCTGGCACCAGACTTG-39), P2fw (59-GATCAGATACCGTCGTAGTC-39), P2rev (59-GACTACGACGGTATCTGATC-39), P3fw (59CAGGTCTGTGATGCCCTTAG-39), P3rev (59-CTAAGGGCATCACAGACCTG-39) were used, to obtain four gene fragments of 350–530 bp for sequencing. Primer locations were 531 (P1fw, P1rev), 995 (P2fw, P2rev) and 1343 bps (P3fw, P3rev) downstream of the 39 end of primer SSU1. We used 1 mL whole-cell DNA and a standard amplification program (30 cycles of 94 C for 1 min, 56 C for 2 min and 72 C for 3 min). Amplification of the 18S rRNA gene fragments was viewed by ethidium bromide staining in a 2% agarose gel electrophoresis. Amplification with two internal primer pairs (P1fw/P2rev, P2fw/P2rev) produced two PCR products, one Thecamoeba band and one Cochlonema band (FIG. 3). According to sequence data Thecamoeba fragments are supposed to be about 80 bp longer than zoopagalean fragments. Bands were separated on a 2% agarose gel and smaller bands were excised. Amplification of flanking fragments produced only one band, which were more likely to represent fungal bands considering the approximate size. A control PCR of uninfected Thecamoeba quadrilineata cultures was performed to rule out other fungal contaminations. Here amplification of the flanking fragments did not lead to a PCR product and amplification of the P1fw/P2rev and P2fw/P2rev fragments produced only one band identical in size to the bigger band produced for infected amoebae. In addition this was confirmed by sequencing and subsequent NCBI-BLAST search, showing highest identities with published fungal sequences. Potential fungal bands were excised with a clean razorblade and purified with the Amersham Pharmacia (Vienna, Austria) purification kitH. Amplified fragments were sequenced directly from the PCR products with the ABI PRISMH Big Dye sequencing kit and a 310 ABI PRISM automated sequencer (Applied Biosystems, Langen, Germany). Sequencing was performed with varying concentrations of the PCR products (1 mL and 3 mL) from at least two independent sample setups. At least two sequences from both strands were obtained from all fragments. Fragments KOEHSLER ET AL: COCHLONEMA EURYBLASTUM 217 subsequently were combined to a single sequence for further processing. Alignment and Cluster analysis.—Multiple sequence alignment was performed by subsequent pairwise alignment with the Clustal X application (Thomson et al 1997). The alignment was imported into the GENEDOC sequence editor (Nicholas et al 1997) and manually refined to obtain a better consensus. For cluster analysis the 18S rDNA sequence of C. euryblastum was aligned with published zoopagalean sequences (Tanabe et al 2000). Primer sites, unique gaps, insertions and ambiguously aligned sites were excluded from the analysis. The dataset for phylogenetic analyses consisted of 1480 ˜ aligned sites. Two representatives of the Kickxellales (AF007542, AF007543) were chosen as outgroup. Cluster analysis was performed by constructing a cladogram with the PHYLIP package (Felsenstein 1989). Analyses were performed with different evolutionary models including neighbor joining analysis of Kimura two-parameter distance estimates, maximum parsimony and maximum likelihood. The confidence of the branching order was assessed by the generation of 1000 bootstrap replicates for all three methods. Consensus trees were generated from the resulting trees with CONSENSE and prepared as a figure with TreeView (Page 1996). Sequence data was deposited in GenBank and is available at accession number No. DQ520640. FIG. 1. Brightfield microscopy of Thecamoeba quadrilineata parasitized by Cochlonema euryblastum; mature stage with large snail-like thallus giving rise to outgrowth of branched hyphae that segregate into conidia beginning from the distal tip. The host amoeba already has died at this advanced stage, but the cell membrane is still intact (arrows). 7503; Tq 5 Thecamoeba quadrilineata, t 5 thallus, hy 5 hyphae, c 5 conidia. RESULTS Cochlonema euryblastum parasitized the Thecamoeba host quickly and effectively. Within 2 d of coculturing hyphae growing out of the meanwhile immotile amoebae were visible (FIG. 1). Ultrathin sections of infected T. quadrilineata show the different developmental stages of C. euryblastum (FIG. 2A–D). Conidia were found outside the amoebae as well as within the cytoplasm of the host after ingestion. Young and intermediate stages (FIG. 2A–C) already contained nuclei and electron transparent vacuoles. Conidia germinated to produce coiled thalli as mature stages (FIG. 2D). When a thallus attained full size it began to produce conidiogenous hyphae, which initially were not segregated into conidia but which broke through the protoplasmic pellicle of the amoeba and then segregated after a considerable period of time in the environment. Of note, spores were not observed in any of the cultures. The 18S rRNA gene of C. euryblastum exhibits a total length of 1777 bp (FIG. 3) with a GC-content of 49.35%. PCR products of Cochlonema euryblastum and Thecamoeba quadrilineata including a control PCR with uninfected amoebae is shown (FIG. 3). The sequence displayed highest similarity to Kuzuhaea moniliformis, a zoopagalean fungus. The calculated sequence similarity between K. moniliformis and C. euryblastum is 84.35%. The phylogenetic analysis with three different treebuilding programs resulted in homologous consensus trees with bootstrap supports of . 50% for all branches (FIG. 4). We have chosen Spiromyces aspiralis and Spiromyces minutus as outgroup because these two isolates have been shown to be a closely related group of the order Zoopagales based on prior 18S rDNA analyses. A previously detected mycoparasitic-zooparasitic clade of Syncephalis depressa, Thamnocephalis sphaerospora and Rhopalomyces elegans (Tanabe et al 2000) forms a sister group with a cluster of Piptocephalis corymbifera, Kuzuhaea moniliformis and C. euryblastum, also representing a mycoparasitic-zooparasitic clade in this study. These groupings are strongly supported by high bootstrap values. Zoophagus insidians exhibits the highest substitution rate within order Zoopagales reflected by its external position. DISCUSSION The isolated fungus was identified morphologically as Cochlonema euryblastum. The general structural traits observed with C. euryblastum are in agreement with 218 MYCOLOGIA FIG. 2. Electron microscopy of Thecamoeba quadrilineata parasitized by Cochlonema euryblastum. A. Conidia are found inside and outside of the host amoeba, two young stages of the parasitic endocytobiont are located within the cytoplasma of the host. B. Host amoeba harboring five young stages of the endocytobiont, a large food vacuole contains numerous freshly ingested bacteria. C. Host amoeba showing two young parasitic stages; the nucleus is characterized by a huge karyosome or endosome, the cytoplasm is packed with food vacuoles. D. Host amoeba with two mature thalli, many nuclei are scattered throughout the cytoplasm together with small vacuoles and vacuoles containing electron-dense bodies; the left side of the parasite is tapering into a more slender frontal part with the initials of a conidiogenous hypha (arrows) containing nuclei; note that the parasite actually is enveloped by a host membrane and that mitochondria are accumulated in the cytoplasmic area between both parasites. A–C: 36003, D: 84003; c5 conidia, cm 5 pellicle of the host amoeba, dv 5 dense bodies, en 5 endosome, fv 5 food vacuole, hy 5 hyphae, mi 5 mitochondria, n 5 nucleus, p 5 parasitic endocytobiont, sv 5 small vacuole, t 5 thallus, Tq 5 Thecamoeba quadrilineata. those described for C. odontosperma (Saikawa and Sato 1991) with the only difference being that the conidia of C. euryblastum have a smooth surface compared to the conidia of C. odontosperma showing protuberances at the cell surface which appear as bumps on the conidia under the light microscope. Unfortunately no sequence data of C. odontosperma are available to date. Another interesting observation was that spores as described for C. euryblastum by Michel and Wylezich (2005) could not be observed in the present study. However this might be the result of parasite-host interactions. Such interactions between amoebae and their endocytobionts, bacteria as well as fungi, are well known. On one hand, amoebae can alter the shape and even the virulence of their endocytobionts and, on the other hand, endocytobionts can alter the morphological and physiological capabilities of their host amoebae (Barker et al 1993, Cirillo et al 1997, Steenbergen et al 2001, Walochnik et al 2005). The hypothesis of host-parasite interactions in the current case is corroborated by the conspicuous accumulation of mitochondria in the area between the two parasites (FIG. 2D), indicating high metabolism in this area. KOEHSLER ET AL: COCHLONEMA EURYBLASTUM 219 FIG. 3. PCR-products of infected (lanes 1–4) and uninfected (lanes 5, 6) Thecamoeba quadrilineata on a 2% agarose gel; Lane 1: SSU1/Pf1rev, Lane 2: Pf1fw/Pf2rev, Lane 3: Pf2fw/Pf3rev, (Lane 2/3: upper lane T. quadrilineata, lower lane C. euryblastum) Lane 4: Pf3fw/SSU2, M: stepmarker, Lane 5: Pf1fw/Pf2rev, Lane 6: Pf2fw/Pf3rev. The order Zoopagales (Zygomycota, Zygomycetes) comprises five families, with all members being obligatory parasites of other fungi or microscopic animals. Ectoparasitic and predacious species are characterized by the development of haustoria invading the host tissue, while endoparasitic species are characterized by an internal thallus without haustorium development. In addition to the family Cochlonemataceae, which represents parasites of different amoebae and nematodes, the Zoopagales include the Zoopagaceae, predators of nematodes, amoebae and rotifers, the Helicocephalidaceae, parasitic on nematode eggs, and the Piptocephalidaceae and Sigmoideomycetaceae, both mycoparasites. Cochlonemataceae either produce an internal thallus without development of a haustorium or an external thallus with haustoria formation only within the host (Benny et al 1992, Hawksworth et al 1995). C. euryblastum was first described by Charles Drechsler (1942), who characterized it as a parasitic fungus destructive to soil amoebae. C. euryblastum conidia are ingested by the amoebic host by phagocytosis and germinate to produce a longish-ovoid thallus in the cytoplasm, which develops into a characteristic coil-shaped thallus. At that stage amoebae become rounded and immotile. The thallus subsequently produces hyphae that branch several times after growing through the host pellicle. The conidiogenous hyphae become fragmented by cross wall formation resulting in a chain of conidia. The fragmented hyphae are fragile and release single conidia, which can be observed near the dead amoebae (Drechsler 1942, Michel 1999, Saikawa and Sato 1991). Cochlonemataceae and Zoopagaceae originally were considered to be one family, until FIG. 4. Neighbor joining tree based on 18S rDNA sequences of different representatives of the Zoopagales. Distance measures were calculated with a Kimura twoparameter correction. Bootstrap values are based on 1000 replicates and are given at the nodes (NJ/MP/ML). Duddington (1973) transferred the parasitic forms to the Cochlonemataceae and left the predacious taxa in the Zoopagaceae. In 1979 Benjamin added the Piptocephalidaceae and the Helicocephalidaceae to this order. The Sigmoideomycetaceae were transferred from the Mucorales to the Zoopagales in 1995 (Hawksworth et al 1995), which was confirmed in 2000, when Chien reported on the formation of haustoria within this family. To date little sequence information is available from this order and there are no sequences of the family Cochlonemataceae available, mainly due to difficulties in culturing these fungi. Tanabe et al (2000) investigated the molecular phylogeny of parasitic zygomycota and sequenced among others six zoopagalean fungi. In their study a monophyletic cluster of Zoopagales, Kickxellaes and Harpellales was supported. Furthermore they identified a monophyletic mycoparasitic-zooparasitic clade within the Zoopagales, consisting of Syncephalis depressa, traditionally classified as Piptocephalidaceae, Rhopalomyces elegans (Helicocephalidaceae) and Thamnocephalis sphaerospora (Sigmoideeomycetaceae). This clade formed a sister group of a clade comprising two Piptocephalidaceae isolates. These findings interfered with traditional classification. Phylogentic analyses in the current study are concordant with Tanabe’s observations with S. depressa 220 MYCOLOGIA clustering with R. elegans and T. sphaerospora. Again two members of the Piptocephalidaceae, namely Kuzuhaea moniliformis and Piptocephalis corymbifera, form a sister group but now including the zooparasitic C. euryblastum. Furthermore, the previously observed external position of Zoophagus insidians was confirmed with all treebuilding methods. It has been shown that the clade of S. depressa, T. sphaerospora and R. elegans is not the only zooparasitic-mycoparasitic clade within the Zoopagales but that K. moniliforms, P. corymbifera (Piptocephalidaceae) and C. euryblastum also represent a clade of zooparasitic and mycoparasitic fungi. Adding C. euryblastum to the phylogenetic tree corroborates Tanabe’s findings that the molecular phylogeny of the order Zoopagales based on the 18S rDNA does not correlate with the traditional classification. It was demonstrated that S. depressa not only clusters with members of other families but also that member of the Cochlonemataceae appears to be more closely related to members of the Piptocephalidaceae. When constructing a second phylogenetic tree with more families of the Zygomycota, also this was concordant with Tanabe’s observations. The Zoopagales, Kickxellaes and Harpellales distinctly formed a monophyletic group supported by high bootstrap values (.70%) in all tree building methods (data not shown). To address questions on the relationships within the order Zoopagales more sequence data clearly are required. Groupings interfering with traditional classification based on morphology (e.g. the zooparasitic and mycoparasitic clades reported in this study and in prior studies) are a major issue, as are highly divergent isolates within the order, such as Zoophagus insidians. Tanabe et al (2004) reported that RPB1 (RNA polymerase II largest subunit) analyses outperform 18S rRNA gene analyses, with RPB1 analyses basically corroborating 18S rRNA gene phylogeny and partly reflecting intra-ordinal relationships within the Zygomycota more accurately. In that study the monophyletic clade of the Zoopagales, Kickxellales and Harpellales was not supported and a closer relationship of Zoopagales with Entomophtohorales and Blastocladiales was revealed. However, in contrast to the 18S rRNA gene analyses, the Zoopagales represented a monophyletic clade in the RPB1 analysis. Nevertheless phylogenetic studies based on either of these loci leave specific questions unanswered and show several inconsistencies. Altogether, our 18S rDNA analysis clearly corroborates the morphological identification of the fungus parasitizing an isolate of Thecamoeba quadrilineata as Cochlonema euryblastum. To our knowledge these are the first sequence data for the Cochlonemataceae and cluster analyses supported the grouping with other zoopagaleans with high bootstrap values. ACKNOWLEDGMENTS The authors thank Susanne Glöckl, Iveta Häfeli and Jacek Pietrzak from the Department of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology, Medical University of Vienna, for technical support. We also thank Gerhild Gmeiner (Laboratory for Electron Microscopy, CIFAFMS, Koblenz; Head: B. Hauröder) for excellent technical assistance. LITERATURE CITED Barker J, Lambert PA, Brown MR. 1993. Influence of intraamoebic and other growth condition on the surface properties of Legionella pneumophila. Infect Immun 61: 3503–3510. Benjamin RK. 1979. Zygomycetes and their spores. In: Kendrick B., ed. The whole fungus: the sexual-asexual synthesis. Vol. 2. Ottawa: National Museums of Canada. 573 p. Benny GL, Benjamin RK, Kirk PM. 1992. A reevaluation of Cunninghamellaceae (Mucorales), Sigmoideomycetaceae fam. nov. and Reticulocephalis gen. nov.: Cladistic analysis and description of two new species. Mycologia 84:615–641. Brief J. 2005. 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