The authors have declared that no competing interests exist.
Conceived and designed the experiments: RS LKM MM. Performed the experiments: RS. Analyzed the data: RS NS MM. Contributed reagents/materials/analysis tools: LKM MM. Wrote the paper: RS LKM MM.
In 2007, a novel, putatively photosynthetic picoeukaryotic lineage, the ‘picobiliphytes’, with no known close eukaryotic relatives, was reported from 18S environmental clone library sequences and fluorescence in situ hybridization. Although single cell genomics later showed these organisms to be heterotrophic rather than photosynthetic, until now this apparently widespread group of pico-(or nano-)eukaryotes has remained uncultured and the organisms could not be formally recognized. Here, we describe
Microbial plankton plays a pivotal role in global biogeochemical processes and provides amenities and services that are essential to mankind’s existence, including food production, remediation of waste and regulation of aspects of the climate in the biosphere
A large fraction of the
Most of these methods have also been applied to a widely distributed group of uncultured picoeukaryotes that represent a deep evolutionary branch without clear affinities to other eukaryotes. Initially described from cold and polar waters using 18S rDNA clone libraries and FISH, as a group of picoeukaryotic phycobilin-containing algae with affinities to cryptophytes and katablepharids and named ‘picobiliphytes’
Here we used a fluorescent mitochondrial marker to isolate single ‘(pico)biliphyte’ cells by FACS and established a first culture of these organisms. A detailed light and electron microscopic study based on this culture allowed us to describe a new genus and species (
About 600 ml of surface seawater (5 m depth) was collected from the North Sea (Helgoland Roads, 54°11′N, 7°54′E, Germany) in August 2008 and successively filtered through 10 µm and 2 µm Isopore membrane filters (Millipore). From the filtrate, 50 ml was centrifuged at 4000
A ‘(pico)biliphyte’-positive enrichment culture was prepared for fluorescence-activated cell sorting by adding 20 nM MitoTracker® Green FM (M7514, Invitrogen) to 10 ml of sample and incubated at 15°C in the dark for 15–20 min. Cells were sorted with a FACSvantageSE (Becton Dickinson, NJ) using an Argon laser at 488 nm. Cells with high green fluorescence and forward scatter were targeted and sorted directly into PCR tubes for further confirmation of ‘(pico)biliphytes’ by PCR amplification and DNA sequencing. Single cell sorting was performed for the targeted region (
1A. Approximately 100 cells from each of three regions with higher fluorescence/forward scatter (R1–R3) were sorted into PCR tubes. Cells marked in ‘red’ in the cytogram corresponded to ‘(pico)biliphytes’ by PCR amplification and sequencing. 1B. Sorted cells used for primary amplification with ‘picobiliphyte’-specific primers (PICOBI01F/P01ITS1R) and reamplified with PICOBI01F and 1055rev. Amplicons in region R1 were confirmed by DNA sequencing to correspond to ‘(pico)biliphytes’. Arrow 650 base pairs; M 1 Kb ladder.
For fluorescence microscopy (FL), cells (from a 10 day old culture) were fixed with 1.25% (v/v, final concentration) glutaraldehyde (GA) in culture medium for 30 min on ice. Fixed cells were directly placed on a glass slide pre-coated with poly L-lysine (P8920, Sigma) and allowed to settle for 30 min at room temperature. For FL, cells were probed with
For transmission electron microscopy, a cell suspension (10 ml) was fixed with a mixture of 0.32% freshly prepared para-formaldehyde (PFA), 0.125% glutaraldehyde (GA) and 0.01% of osmium tetroxide (v/v, final concentration), and incubated on ice for 30 min. The fixed cells were subsequently transferred to 1.5 ml centrifuge tubes (pre-coated with dichlorodimethylsilane (40410, Fluka, Buchs, Switzerland)) and pelleted at 4000
For scanning electron microscopy; cells were fixed with 1% PFA and 1.25% GA (v/v) for 30 min in culture medium. Fixed cells were directly placed on a glass slide, coated with poly L-lysine and allowed to settle for 30 min. Cells were gradually washed to decrease the salinity with 100%, 50% and 25% (v/v) FSSW followed by dehydration as in Martin-Cereceda et al. (2009) except that the last critical point drying step was with liquid CO2
The
Overall 191 environmental picozoan 18S rDNA sequences retrieved from the Genbank database (
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Seawater samples collected between July and October in 2007, and August 2008 from Helgoland Roads (Germany) and subsequently filtered through 10 µm and 2 µm membrane filters, contained DNA from ‘(pico)biliphytes’ as demonstrated by PCR amplicons using primers specific for ‘(pico)biliphytes’ (results not shown). ‘(Pico)biliphyte’- positive samples were regularly transferred into 0.1 µm filter-sterilized seawater (FSSW) and the presence of ‘(pico)biliphytes’ monitored by PCR and DNA sequencing. These ‘enrichments’ were used for flow cytometry and sorting. We employed a mitochondrial marker (MitoTracker® Green FM) and obtained a single cell-derived culture verified by rDNA sequencing using ‘(pico)biliphyte’-specific and universal eukaryotic ribosomal DNA (rDNA) probes (
The cells of
2A. Differential interference contrast of a chemically fixed cell. Inset shows phase contrast image of a live cell from tissue culture flask photographed with an inverted microscope (Scale bar 5 µm). 2B. Fluorescence and phase contrast overlay, nucleus (blue), mitochondrion (red). 2C. SEM image. 2D. A longitudinal section through a cell in the plane of the flagella, viewed from the cell’s left. 2E. A 3 D serial section reconstruction of the cell depicted in 2D. AF/PF (anterior−/posterior flagellum); AP/PP (anterior/posterior part of the cell); G (Golgi body); M (mitochondrion); MB (‘microbody’); N (nucleus); tr1,tr2 (distal [tr2] and proximal [tr1] flagellar transitional regions); P (posterior digestive body); Cl (cleft separating the anterior from the posterior part of the cell); vc (vacuolar cisterna).
The cells of
The ultrastructure of
The single mitochondrion with tubular cristae (
Single mitochondrion with tubular cristae, membrane bound (single and double) electron dense material (edsm1 and 2 respectively) and regular projections of the outer envelope membrane. 3A. a double membrane bound edsm2 near the ventral surface; serial sections (1–4) of the edsm2 displayed some tube-like structures in the lumen (thin lines); also note that the edsm2 is a branched structure. 3B. A membrane invagination of edsm2 into the mitochondrion that reveals continuity between the mitochondrial envelope membranes and the two membranes encircling the edsm2. 3C. Oblique section of a cell from dorsal right to ventral left with edsm1 and edsm2 displayed. The edsm1 (on the left) is positioned between the outer (black arrow) and inner (white arrow) mitochondrial envelope membrane and the edsm2 (on the right) with a double membrane (higher magnifications of the two edsms on the right. A large number of short cylindrical membrane protrusions termed ‘mitovilli’ (arrow heads) extend from the outer mitochondrial membrane towards posterior digestive body (P), they terminate in granular material covering the posterior digestive body. A vacuolar cisterna (vc) in the ‘cleft’ region separates the anterior from posterior part of the cell and is only absent (i.e. contains a large hole) in the area of the mitovilli (3C). Towards the right are shown higher magnifications of two serial sections from the cell depicted in 3C revealing details of the mitovilli-posterior digestive body junction. F (feeding apparatus with basket fibers; G (Golgi body); M (mitochondrion); N (nucleus); P (posterior digestive body); vc (vacuolar cisterna). Numbers at the top right indicate the section number of a series. Scale bar: 100 nm.
There are two membrane-bound inclusions with electron-dense, granular contents located at specific positions inside the mitochondrion (edsm1, edsm2,
The lower side of the mitochondrion adjacent to the posterior part of the cell (the bottom of the seating area) displayed another highly unusual specialization. Over an area of ∼ 1×0.6 µm (
The single Golgi body is located between mitochondrion, nucleus and basal body of the anterior flagellum closely associated (resting on) with the seating area of the mitochondrion (
4A. Longitudinal sections of a
Two large, spherical microbodies (MB; with granular contents, bounded by a single membrane) of 600–800 nm diameter are located in the dorsal region of the anterior part of the cell, besides the mitochondrion (
The anterior part of the cell is separated from the posterior part by a large, horizontally-oriented, plate-like vacuolar cisterna (vc,
The posterior part of the cell consists of numerous vesicles and vacuoles of different sizes and electron density but lacks ribosomes and endoplasmic reticulum, which are confined to the anterior part. In addition it contains the cytostome and feeding basket (see below). The lengths (and volumes) of the posterior part (PP) were found to vary considerably among sectioned cells (from anterior to posterior: 0.6–1.5 µm, compare e.g.
The first structure is a large vacuole (“posterior digestive body,” P) that is located in the ventral region of the posterior part of the cell adjacent to the vacuolar cisterna and the mitovilli (
The second conspicuous structure in the posterior part (PP) of the cell is the cytostome/feeding basket that together comprise the feeding apparatus (‘F’,
The two flagella emerge from the ventral cell surface of the cell, close to the junction of the Golgi body and the ventral tip of the mitochondrion (
5A. Longitidinal section of
6A. Electron micrograph shows consecutive serial sections of
7A. Non-consecutive serial sections from dorsal to ventral (sections are oblique to cell’s right). Each basal body is connected to two microtubular flagellar roots. The anterior root 1 (Ar1) runs anteriorly to cell’s left. The Ar2 runs posteriorly, at the right side of the cell and passes the cleft (cl). The other two flagellar roots originate from the posterior basal body and extend towards the posterior part of the cell. One of the posterior flagellar roots (Pr1) runs on the left side of the cell. The other broader posterior flagellar root (Pr2), runs between the Ar1 and Pr1. 7B. The Pr2 with 6 microtubules (arrowheads) obliquely sectioned. 7C. A cell with the Pr2 passing the vacuolar cisterna and mitochondrion. 7D. Consecutive serial cross sections through the Pr2 located in a depression of the mitochondrion. AF/PF (anterior−/posterior flagellum); Ab/Pb (anterior−/posterior basal body); Ar1/Ar2 (Anterior microtubular flagellar roots 1 and 2); Pr1/Pr2 (posterior microtubular flagellar roots 1 and 2); G (Golgi body); M (mitochondrion); vc (vacuolar cisterna); P (posterior digestive body); CMT (secondary cytoplasmic microtubule). Numbers at the top right indicate the number of the serial section. Scale bar: 200 nm.
The basal body and the flagellar transitional region display several unusual features: Basal bodies are relatively short (360–380 nm) and most of their lumen is filled with electron dense material (
During interphase, the basal bodies generally do not appear to be associated with probasal bodies, but in one cell (likely in preparation for cell division), we observed probasal bodies associated with each basal body (
Serial sections revealed the presence of four microtubular flagellar roots, two associated with each basal body. We have given the roots descriptive terms (Ar1, Ar2, Pr1, Pr2;
The cells are covered only by the plasma membrane with no scales or glycocalyx being discernible. Often, rod-shaped bacteria were encountered apparently physically attached to the plasma membrane by their ends. These bacteria were (ultra)structurally intact and could associate with any part of the plasma membrane (except flagella) (not shown).
A molecular phylogenetic analysis of a broadly sampled taxon set (104 taxa of eukaryotes, excluding only Excavata) using a data set consisting of 18S rDNA, 5.8S rDNA and 28S rDNA (4461 aligned characters) could not position the Picozoa in one of the known eukaryotic supergroups. Neither monophyly of the Hacrobia
The phylogeny is based on 1253 aligned characters of the SSU rDNA and includes 201 sequences of Picozoa. Most sequences are database entries derived from clone libraries (nine environmental sequences generated from a sample taken at Helgoland Roads and one sequence from
Heterotrophic, marine protists of picoplanktonic size (cells may pass through a 3 µm membrane filter) mostly characterized by either of two signature sequences in the nuclear-encoded SSU rDNA, 5′
With the characteristics of the phylum.
With the characters of the phylum. Taxa are characterized by the signature sequence 5′
With the characters of the order. The most inclusive clade containing taxa with the sequence accession numbers HQ868687 (unresolved), EU368015 (clade P1), EU368029 (unresolved), DQ222877 (clade P2) and
Cells are biflagellate with a long and a short flagellum inserted laterally. Each cell consists of two nearly hemi-spherical parts separated by a cleft. The anterior part contains the flagellar apparatus, nucleus, endoplasmic reticulum, single Golgi body and mitochondrion with tubular cristae, whereas the posterior part is variable in size and contains the feeding apparatus and numerous vesicles and vacuoles. The anterior and posterior parts of the cell are separated by a single, large, vacuolar cisterna that leaves only part of the mitochondrion in direct contact with the posterior part. The cells exhibit a unique mode of motility: After extended periods of rest, a stereotypic pattern is initiated consisting of a rapid, short-distance jump, immediately followed by a slower, dragging cell movement in the opposite direction. This pattern may be repeated several times before cells finally show an extremely fast and extended movement away from their original position (termed ‘skedaddle’). The genus represents the most inclusive clade containing the type species,
Characters of the genus. The oblong cells vary in length between 2.6–3.8 µm, their width is 2–2.5 µm. The longer flagellum measures 12–14 µm; the shorter flagellum 7–9 µm. The longer (anterior) flagellum is oriented towards the anterior; the shorter (posterior) flagellum towards the posterior end of the cell. The nucleus is hemispherical, and over the spherical part of its surface is appressed to the plasma membrane. The feeding apparatus essentially consists of a basket of about 50–60 parallel running fibers (30 nm repeat) of varying lengths that extend from the ventral surface where they are attached to the plasma membrane for up to 1.2 µm towards the dorsal part where they terminate. The basket is open towards the cell’s anterior and dorsal parts, but closed towards the posterior end of the cell. The slit-like cytostome is formed at the ventral cell surface where the ‘side walls’ of the basket fibers connect to the plasma membrane and extends in the anterior-posterior direction for a length of about 1 µm, its width being about 150 nm.
Marine plankton.
Surface water (5 m depth) from Helgoland Roads, (54°11′N, 7°54′E), North Sea, Germany.
The name-bearing hapantotype is a block of resin-embedded cells for electron microscopy (prepared from a single cell-derived culture established from the original natural sample) deposited at the Culture Collection of Algae at the University of Cologne (CCAC;
Named after its unique stereotypic mode of cell motility, which consists, in succession, of a short fast jump (ju-) into the anterior direction, a slow drag (-dra-) into the opposite direction and an extremely fast and extended movement of the cell away from its original position (skedaddle; -skeda). Although colloquial in the English language and of unknown origin, the name ‘skedaddle’ may be derived from the Greek σκεδασμός (skedasmos,“dispersion”).
The heterotrophic protist
We feel that these features together with their unresolved position in the eukaryotic phylogenetic tree justify the recognition of this widespread group of marine pico- or nanoplanktonic protists at the phylum level. We recognize, however, that we have investigated only one representative species and don’t know to what extent the structural features described for
Picoplankton was originally defined as those organisms whose cell size lies between 0.2 and 2 µm
What do our results contribute to the question of whether Picozoa are picoeukaryotes or not or phrased differently, what can we conclude about the ‘real’ dimensions of these cells? First, we can conclude that not only DNA but live cells of Picozoa can pass through 2 µm filters since we established a culture of
Whereas the heterotrophic nature of the Picozoa is now beyond doubt, their mode of feeding remains essentially unknown. Kim et al. (2011) speculated that phycoerythrin fluorescence in Picozoa may have been the result of phagotrophic feeding of Picozoa on cyanobacteria, e.g.
Do our electron microscope observations shed light on the feeding behavior and the likely food source of the Picozoa? One of the most unusual structural features of
Could Picozoa perhaps feed on viruses as well as suggested by Yoon et al. (2011)? Viruses constitute the most abundant group of nucleic acid-containing particles in the ocean and up to 108 virus particles per milliliter have been recorded in productive coastal surface waters
During the last decade, culture-independent molecular surveys based on rDNA clone libraries, phylogenetic analyses, and fluorescence
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RS wishes to thank the Alfred-Wegener-Institute for Polar and Marine Research (Bremerhaven, Germany) for hosting initial experiments and the RRZK of the University of Cologne for providing computational resources (CHEOPS).