Published in Journal of Eukaryotic Microbiology, Vol. 64, Issue 2, 2017, p. 257-265 which should be used for any reference to this work 1 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library Mycamoeba gemmipara nov. gen., nov. sp., the First Cultured Member of the Environmental Dermamoebidae Clade LKM74 and its Unusual Life Cycle Quentin Blandeniera, Christophe V.W. Seppeya, David Singera, Michele Vlimantb, Anaele€ Simonc, Clement Duckerta & Enrique Laraa a Laboratory of Soil Biodiversity, Institute of Biology, University of Neuchatel,^ Emile Argand 11, Neuchatel^ 2000, Switzerland b Laboratory of Ecology and Evolution of Parasites, Institute of Biology, University of Neuchatel,^ Emile Argand 11, Neuchatel^ 2000, Switzerland c Laboratory of Microbiology, Institute of Biology, University of Neuchatel,^ Emile Argand 11, Neuchatel^ 2000, Switzerland Keywords ABSTRACT Budding; Discosea; eukaryotic diversity; fungus; high throughput sequencing; Since the first environmental DNA surveys, entire groups of sequences called Longamoebia; ribosomal genes; serial “environmental clades” did not have any cultured representative. LKM74 is an dilution; yeast. amoebozoan clade affiliated to Dermamoebidae, whose presence is perva- sively reported in soil and freshwater. We obtained an isolate from soil that Correspondence we assigned to LKM74 by molecular phylogeny, close related to freshwater Q. Blandenier, Laboratory of Soil Biodiver- clones. We described Mycamoeba gemmipara based on observations made sity, Institute of Biology, University of with light- and transmission electron microscopy. It is an extremely small Neuchatel,^ Emile Argand 11, Neuchatel^ amoeba with typical lingulate shape. Unlike other Dermamoebidae, it lacked 2000, Switzerland ornamentation on its cell membrane, and condensed chromatin formed charac- Telephone number: +41-32-718-23-27; teristic patterns in the nucleus. M. gemmipara displayed a unique life cycle: FAX number: +41-32-718-30-01; trophozoites formed walled coccoid stages which grew through successive e-mail: [email protected] buddings and developed into branched structures holding cysts. These struc- tures, measuring hundreds of micrometres, are built as the exclusive product of osmotrophic feeding. To demonstrate that M. gemmipara is a genuine soil inhabitant, we screened its presence in an environmental soil DNA diversity survey performed on an experimental setup where pig cadavers were left to decompose in soils to follow changes in eukaryotic communities. Mycamoeba gemmipara was present in all samples, although related reads were uncom- mon underneath the cadaver. OUR vision of protist diversity has been radically chal- role in the oceanic microbial loop (Azam et al. 1983). In lenged since the introduction of observation-independent deeper waters, a particular group of excavates, the environmental DNA surveys. Large and deep-branching diplonemids revealed an immense diversity (Lara et al. groups of eukaryotes have been discovered with classical 2009; Lukes et al. 2015). New massive sequencing tech- cloning/sequencing strategies, thus overtaking our estima- nologies also revealed a large diversity in opisthokonts (del tions on eukaryotic environmental diversity. Unsuspected Campo et al. 2015). alveolate clades appeared to be extremely diverse in mar- Other systems like soils, however, have been by far not ine systems (Lopez-Garc ıa et al. 2001), and were found as deeply studied as the ocean. The high prevalence of later to be exclusively composed by parasitoids (Guillou fungal, plant, and metazoan sequences has been for a et al. 2008). Likewise, many new stramenopile lineages long time a major hindrance for studies on soil protist were discovered in the early 2000s in marine systems diversity (Lesaulnier et al. 2008). Still, previously unsus- (Massana et al. 2004). These organisms, which include pected deep branching clades have also been discovered nowadays 25 different lineages spread all across the tree in soils, like the Opisthosporidia (also known as Rozel- of stramenopiles, were found to be the most diverse and lomycota) (Karpov et al. 2013; Lara et al. 2010). Recently, numerous bacterivores in the sunlit part of oceans (Mas- the development of high throughput sequencing has sana et al. 2014), and thus vesting them with a prominent allowed obtaining high numbers of phylotypes, showing 2 promising results in terms of overall microeukaryotic diver- Page’s Amoeba Saline medium amended with 1 g per sity (Geisen et al. 2015). Relationships among organisms 100 ml of Tryptone Soy Broth and Escherichia coli as food have been inferred using sound experimental designs and organism. Amoebae were subcultured regularly to obtain approaches such as co-occurrence networks (Lentendu pure, monoprotistan strains and to lower the proportion of et al. 2014). However, strong conclusions on the organ- environmental bacteria. Cultures were kept at 12 °C. isms’ morphology and function can only be provided by Cultures were observed using Utermohl’s€ plankton direct observation or, even better, culturing. Therefore, a chambers with an inverted microscope (Olympus IX81). current challenge in eukaryotic microbiology is to identify Cells were measured at different life stages, which were the organisms hiding behind these environmental clades morphologically documented and photographed with light and to infer their ecological function. For this purpose sev- microscopy. Different life stages were also documented eral approaches have been used. Fluorescence in situ using Methyl blue (C37H27N3Na2O9S3) to stain cell walls hybridization (FISH) in combination with scanning electron and condensed cytoplasm. microscopy has been recently applied to the characteriza- We also used a full flask containing active and coccoid tion of Paulinellida (Euglyphida testate amoebae) living in life stages for transmission electron microscopy (TEM). In forest litter (Tarnawski and Lara 2015). Bulk soil protists that purpose, we pelleted cells from a thriving culture. Fix- remain, however, widely inaccessible to FISH probes ation, staining, and mounting were achieved as described because of the large amount of soil particles unless organ- in (Lara et al. 2006). Observations were made on a Philips isms are large enough to be isolated individually (e.g. cili- CM 100 transmission electron microscope. ates, macroscopic mycetozoa, and testate amoebae). Typically, naked amoebozoans are numerous in soils (Gei- Molecular analyses sen et al. 2015), and harbour a wide array of lifestyles and morphologies (Shadwick et al. 2009). In addition, a flask containing 10 ml of a thriving culture The amoebozoan environmental clade LKM74, named was used for DNA extraction. Cells were removed from after the first clone encountered in an environmental DNA the flask bottom with a cell scraper, and the resulting survey (van Hannen et al. 1999) is quite abundant and well supernatant was placed into a Falcon tube and centrifuged distributed in soils (Corsaro and Venditti 2013), but also at maximum speed during 20 min. The obtained pellet present in freshwaters (Di Filippo et al. 2015; Richards was placed into 200 ll of Guanidine thiocyanate buffer et al. 2005) and peat bogs (Lara et al. 2011). This clade and nucleic acids were extracted following a protocol has been repeatedly placed in the vicinity of Dermamoeba (Chomczynski and Sacchi 1987) adapted after (Lara et al. algensis in small subunit ribosomal gene trees (18S rRNA) 2007). We amplified the 18S rRNA gene using primers EK (Corsaro and Venditti 2013; Kudryavtsev and Pawlowski 82F (GAAACTGCGAATGGCTC) and EK 1498R (CACC- 2015), although this relationship remained weakly sup- TACGGAAACCTTGTTA) in a total volume of 30 ll with an ported. Despite its pervasive presence in many environ- amplification profile consisting of 4 min at 95 °C followed ments, the organisms have never been kept in culture and by 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 1 min their morphology remains unknown. In this study, we 30 s at 72 °C with a final elongation of 10 min at 72 °C. describe a tiny naked amoeba isolated from the bulk soil Sequencing was carried out using a BigDyeTM Terminator of a coniferous forest by serial dilutions. We affiliated it to Cycle Sequencing Ready Reaction Kit (PE Biosystems, LKM74 based on 18S rRNA gene sequences, character- Geneve, Switzerland) and analysed with a ABI-3130XL ized its complex life cycle and feeding strategy, and docu- DNA sequencer ABI PRISM 3700 DNA Analyzer (PE mented its ultrastructure. Furthermore, we demonstrated Biosystems). The sequence was deposited in GenBank that this species is a typical soil inhabitant by following with the following accession number: KX687875. related sequence reads in an environmental eukaryotic DNA survey of soils. This study was conducted in an Phylogenetic analysis experimental setup where pig cadavers were left to decompose and samples were taken at regular intervals The obtained sequence was placed in an alignment contain- to follow modifications of the microbial eukaryotic commu- ing various sequences from Discosea (with an emphasis on nities in the underlying soil during the process of decay. Longamoebia as defined in Smirnov et al. 2011b) derived either from isolated cells or cultures, or from environmental clone sequences. The root was placed on Vannellida. The MATERIALS AND METHODS alignment is available from the authors upon request. We build a maximum likelihood phylogenetic tree using the Sample