Synthesis of the rosette-inducing factor RIF-1 and analogs.

Studies on the origin of animal multicellularity have increasingly focused on one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta. Single cells of S. rosetta can develop into multicellular rosette-shaped colonies through a process of incomplete cytokinesis. Unexpectedly, the initiation of rosette development requires bacterially produced small molecules. Previously, our laboratories reported the planar structure and femtomolar rosette-inducing activity of one rosette-inducing small molecule, dubbed rosette-inducing factor 1 (RIF-1), produced by the Gram-negative Bacteroidetes bacterium Algoriphagus machipongonensis. RIF-1 belongs to the small and poorly explored class of sulfonolipids. Here, we report a modular total synthesis of RIF-1 stereoisomers and structural analogs. Rosette-induction assays using synthetic RIF-1 stereoisomers and naturally occurring analogs defined the absolute stereochemistry of RIF-1 and revealed a remarkably restrictive set of structural requirements for inducing rosette development.

Choanoflagellate and choanocyte collar-flagellar systems and the assumption of homology.

The similarities between the choanoflagellates and the choanocytes of sponges have been discussed for more than a century yet few studies allow a direct comparison of the two. We reviewed current knowledge of the collar and flagellum and compared their structure and function in the choanoflagellate Monosiga brevicollis and the sponge Spongilla lacustris. Collar microvilli were of similar length and number, but the shape of the collar differed between the two cells. In Monosiga, collars were flared and microvilli were joined by a single band of glycocalyx mid-way along their length; in Spongilla, collars formed a tube and microvilli were joined by a mesh of glycocalyx. Monosiga flagella beat at least four times faster than those in Spongilla. Flagellar vanes were found in both cell types. In both cells, the flagella and so probably also the vanes maintained moving points of contact with the microvilli, which suggested that collars and flagella were integrated systems rather than independent units. There were fundamental differences in how the collar and flagella interacted, however. In Spongilla, the flagellum bent upon contact with the collar; the flagellar amplitude was fitted to the collar diameter. In Monosiga, the flagellar amplitude was unaffected by the collar; instead the collar diameter appeared fitted to the flagellum. These differences suggest that though choanocytes and choanoflagellates are similar, homology cannot be taken for granted. Similarities in collar-flagellum systems separated by 600 million years of evolution, whether maintained or convergent, suggest that these form important adaptations for optimizing fluid flow through micro-scale filters.

Primordial neurosecretory apparatus identified in the choanoflagellate Monosiga brevicollis.

SNARE protein-driven secretion of neurotransmitters from synaptic vesicles is at the center of neuronal communication. In the absence of the cytosolic protein Munc18-1, synaptic secretion comes to a halt. Although it is believed that Munc18-1 orchestrates SNARE complexes, its mode of action is still a matter of debate. In particular, it has been challenging to clarify the role of a tight Munc18/syntaxin 1 complex, because this interaction interferes strongly with syntaxin's ability to form a SNARE complex. In this complex, two regions of syntaxin, the N-peptide and the remainder in closed conformation, bind to Munc18 simultaneously. Until now, this binary complex has been reported for neuronal tissues only, leading to the hypothesis that it might be a specialization of the neuronal secretion apparatus. Here we aimed, by comparing the core secretion machinery of the unicellular choanoflagellate Monosiga brevicollis with that of animals, to reconstruct the ancestral function of the Munc18/syntaxin1 complex. We found that the Munc18/syntaxin 1 complex from M. brevicollis is structurally and functionally highly similar to the vertebrate complex, suggesting that it constitutes a fundamental step in the reaction pathway toward SNARE assembly. We thus propose that the primordial secretion machinery of the common ancestor of choanoflagellates and animals has been co-opted for synaptic roles during the rise of animals.

Cell differentiation and morphogenesis in the colony-forming choanoflagellate Salpingoeca rosetta.

It has been posited that animal development evolved from pre-existing mechanisms for regulating cell differentiation in the single celled and colonial ancestors of animals. Although the progenitors of animals cannot be studied directly, insights into their cell biology may be gleaned from comparisons between animals and their closest living relatives, the choanoflagellates. We report here on the life history, cell differentiation and intercellular interactions in the colony-forming choanoflagellate Salpingoeca rosetta. In response to diverse environmental cues, S. rosetta differentiates into at least five distinct cell types, including three solitary cell types (slow swimmers, fast swimmers, and thecate cells) and two colonial forms (rosettes and chains). Electron microscopy reveals that cells within colonies are held together by a combination of fine intercellular bridges, a shared extracellular matrix, and filopodia. In addition, we have discovered that the carbohydrate-binding protein wheat germ agglutinin specifically stains colonies and the slow swimmers from which they form, showing that molecular differentiation precedes multicellular development. Together, these results help establish S. rosetta as a model system for studying simple multicellularity in choanoflagellates and provide an experimental framework for investigating the origin of animal multicellularity and development.

Ecologically relevant choanoflagellates collected from hypoxic water masses of the Baltic Sea have untypical mitochondrial cristae.

BACKGROUND: Protist communities inhabiting oxygen depleted waters have so far been characterized through both microscopical observations and sequence based techniques. However, the lack of cultures for abundant taxa severely hampers our knowledge on the morphology, ecology and energy metabolism of hypoxic protists. Cultivation of such protists has been unsuccessful in most cases, and has never yet succeeded for choanoflagellates, even though these small bacterivorous flagellates are known to be ecologically relevant components of aquatic protist communities. RESULTS: Quantitative data for choanoflagellates and the vertical distribution of Codosiga spp. at Gotland and Landsort Deep (Baltic Sea) indicate its preference for oxygen-depleted zones. Strains isolated and cultivated from these habitats revealed ultrastructural peculiarities such as mitochondria showing tubular cristae never seen before for choanoflagellates, and the first observation of intracellular prokaryotes in choanoflagellates. Analysis of their partial 28S rRNA gene sequence complements the description of two new species, Codosiga minima n. sp. and C. balthica n. sp. These are closely related with but well separated from C. gracilis (C. balthica and C. minima p-distance to C. gracilis 4.8% and 11.6%, respectively). In phylogenetic analyses the 18S rRNA gene sequences branch off together with environmental sequences from hypoxic habitats resulting in a wide cluster of hypoxic Codosiga relatives so far only known from environmental sequencing approaches. CONCLUSIONS: Here, we establish the morphological and ultrastructural identity of an environmental choanoflagellate lineage. Data from microscopical observations, supplemented by findings from previous culture-independent methods, indicate that C. balthica is likely an ecologically relevant player of Baltic Sea hypoxic waters. The possession of derived mitochondria could be an adaptation to life in hypoxic environments periodically influenced by small-scale mixing events and changing oxygen content allowing the reduction of oxygen consuming components. In view of the intricacy of isolating and cultivating choanoflagellates, the two new cultured species represent an important advance to the understanding of the ecology of this group, and mechanisms of adaptations to hypoxia in protists in general.

A Needle in the Haystack - Mapping Sequences to Morphology Exemplified by the Loricate Choanoflagellate Enibas thessalia sp. nov. (Acanthoecida, Acanthoecidae).

Environmental sequencing surveys unveil an unexpected magnitude of protist biodiversity and help to understand environmental community structure as well as biogeographical patterns. The interpretation of these data is still hindered by the lack of a verified and reliable reference database, which is the important basis for all analyses. References should rely on detailed and valid taxonomical descriptions including both morphology and autecological properties. In fact, obtaining such data is still a major challenge as cultivation-based approaches are very selective. In the present study, we highlight the potential to resample habitats which showed phylogenetically interesting sequences from environmental molecular surveys. We have been able to reveal a choanoflagellate species with the use of a single cell isolation approach in order to achieve a morphological description to the target sequence. This new species, Enibas thessalia sp. nov. now extends a recently described monospecific genus. In addition, we illustrate a nudiform lorica reproduction of the genus Enibas by observation of living cells. The genus belongs to the family of Acanthoecidae, which comprises five genera. The morphology of the genus Enibas shows a striking resemblance to the genus Stephanoeca, which belongs to the other family of loricate choanoflagellates, the Stephanoecidae, indicating that morphology alone might not reflect phylogenetic relations. We demonstrate that mapping sequences to a taxonomical description of species is a valuable tool to verify the organism behind an environmental amplicon. We emphasize the urgent need of integrative taxonomy matching molecular data with morphological features to verify the outcome of phylogenetic analyses.

Biosilicification of loricate choanoflagellate: organic composition of the nanotubular siliceous costal strips of Stephanoeca diplocostata.

Loricate choanoflagellates (unicellular, eukaryotic flagellates; phylum Choanozoa) synthesize a basket-like siliceous lorica reinforced by costal strips (diameter of approximately 100 nm and length of 3 µm). In the present study, the composition of these siliceous costal strips is described, using Stephanoeca diplocostata as a model. Analyses by energy-dispersive X-ray spectroscopy (EDX), coupled with transmission electron microscopy (TEM), indicate that the costal strips comprise inorganic and organic components. The organic, proteinaceous scaffold contained one major polypeptide of mass 14 kDa that reacted with wheat germ agglutinin. Polyclonal antibodies were raised that allowed mapping of the proteinaceous scaffold, the (glyco)proteins, within the costal strips. Subsequent in vitro studies revealed that the organic scaffold of the costal strips stimulates polycondensation of ortho-silicic acid in a concentration- and pH-dependent way. Taken together, the data gathered indicate that the siliceous costal strips are formed around a proteinaceous scaffold that supports and maintains biosilicification. A scheme is given that outlines that the organic template guides both the axial and the lateral growth of the strips.

Loricate choanoflagellates (Acanthoecida) from warm water seas. VII. Calotheca Thomsen and Moestrup, Stephanacantha Thomsen and Syndetophyllum Thomsen and Moestrup.

The tectiform loricate choanoflagellate genera Calotheca, Stephanacantha and Syndetophyllum have all been first described from warm water habitats and share the presence of flattened and often elaborate costal strips in the lorica. The current reinvestigation does confirm both the widespread occurrence of these taxa within the global warm water belt, and largely corroborates the established genus and species matrix. We describe here Stephanacantha oceanica sp. nov. which closely resembles S. campaniformis, and transfer Parvicorbicula zigzag to the genus Stephanacantha, despite differences in costal strip morphology, but based on a complete agreement in lorica constructional details.

Hydrodynamic functionality of the lorica in choanoflagellates.

Choanoflagellates are unicellular eukaryotes that are ubiquitous in aquatic habitats. They have a single flagellum that creates a flow toward a collar filter composed of filter strands that extend from the cell. In one common group, the loricate choanoflagellates, the cell is suspended in an elaborate basket-like structure, the lorica, the function of which remains unknown. Here, we use Computational Fluid Dynamics to explore the possible hydrodynamic function of the lorica. We use the choanoflagellate Diaphaoneca grandis as a model organism. It has been hypothesized that the function of the lorica is to prevent refiltration (flow recirculation) and to increase the drag and, hence, increase the feeding rate and reduce the swimming speed. We find no support for these hypotheses. On the contrary, motile prey are encountered at a much lower rate by the loricate organism. The presence of the lorica does not affect the average swimming speed, but it suppresses the lateral motion and rotation of the cell. Without the lorica, the cell jiggles from side to side while swimming. The unsteady flow generated by the beating flagellum causes reversed flow through the collar filter that may wash away captured prey while it is being transported to the cell body for engulfment. The lorica substantially decreases such flow, hence it potentially increases the capture efficiency. This may be the main adaptive value of the lorica.

Loricate choanoflagellates (Acanthoecida) from warm water seas. III. Acanthocorbis Hara and Takahashi and Stephanoeca Ellis.

A large-scale investigation of warm water loricate choanoflagellate communities has revealed the presence of close to 80 species, which is approximately half of all loricate choanoflagellate taxa described when including also hitherto undescribed forms known to us. We are in the process of stepwise providing a monographic treatment of these communities. The overall aim is to contribute the best possible account of species diversity, based on traditional light and electron microscopical techniques, as a tool for future identification work based on microscopy, and in support of the work in progress with establishing a quality assured molecular tool for future recognition of diversity. In this paper we summarize our findings of species of Acanthocorbis and Stephanoeca, which include the description of several new taxa: A. conicella sp. nov., A. gladiella sp. nov., S. broomia sp. nov., S. naja sp. nov., and S. andemanica sp. nov.

Loricate choanoflagellates (Acanthoecida) from warm water seas. IV. Cosmoeca Thomsen.

Aided by an extensive collection of specimens from warm water seas, it has been possible to revisit the loricate choanoflagellate genus Cosmoeca. While the type species C. norvegica and also C. ventricosa sensu stricto have been described from temperate North Atlantic realms and share a cosmopolitan distribution, the remaining species and morphotypes of Cosmoeca are largely confined to warmer waters. The new data broadly validates the initial circumscription of species of Cosmoeca. The persisting taxonomic puzzle with respect to C. ventricosa, which in addition to the core type accommodates no less than five different morphotypes (form A-E), has been further elucidated. The Cosmoeca paper is part of a 'monographic' series of warm water loricate choanoflagellate contributions in progress, where the aim is to provide the best possible account of warm water species diversity, based on traditional light and electron microscopical techniques, as a tool for future identification work based on microscopy, and in support of the work in progress with establishing a quality assured molecular tool for future recognition of diversity.

Four new choanoflagellate species from extreme saline environments: Indication for isolation-driven speciation exemplified by highly adapted Craspedida from salt flats in the Atacama Desert (Northern Chile).

With this study we aim to extend the knowledge on the biogeography of craspedid choanoflagellates with additional data from extreme environments. Up to now, very little is known about choanoflagellates from extreme saline environments, as most studies have focused on marine and freshwater habitats. Though previously investigated high saline ice biota communities have indicated a possible adaptation to environments with high salt concentrations. Hypersaline endorheic basins, so-called salt flats or salares from the Atacama Desert in Northern Chile provide an intense environment regarding fluctuating and extreme salinities, which allow for studies on evolutionary adaptations of protists to hypersaline conditions. This study focused on choanoflagellate species isolated from different salt flats, their morphological characteristics using light and electron microscopy, molecular marker genes (SSU and LSU rDNA) and their salinity tolerance. Here, we described four new craspedid choanoflagellate species, highly adapted to the hypersaline environment of the Atacama Desert. This study extends our knowledge on choanoflagellate phylogeny and ecology and can become the basis for further molecular studies to understand the mechanisms of adaptations. Additionally, we emphasize the need of adding additional data such as autecological characteristics to amend species definitions, which is only possible from cultivated strains. This data would support the use of molecular data originating from metagenomic analyses also in an ecological context.

A phylogenetic and morphological re-investigation of Diaphanoeca spiralifurca, Didymoeca elongata and Polyoeca dichotoma (Acanthoecida/Choanomonadida) from the Caribbean Sea.

Acanthoecid choanoflagellates are described and classified according to their distinct siliceous lorica, a unique morphological characteristic within protistan species. Only recently it has become clear that beside morphological and developmental data on the lorica molecular data, mainly based on SSU rDNA, it is necessary to resolve phylogenetic relationships within this group. Previously Didymoeca elongata was thought to be an endemic species from Taiwanese waters. However, this species has now been found in the Caribbean Sea at several sampling sites along with two other species, Diaphanoeca spiralifurca and Polyoeca dichotoma. By sequencing the SSU rDNA and other genes and by adding light and electron microscopical data to the description of these species, the taxonomy and phylogeny of Acanthoecida is being better understood.

Extended phylogeny of the Craspedida (Choanomonada).

Currently choanoflagellates are classified into two distinct orders: loricate Acanthoecida and non-loricate Craspedida. The morphologically based taxonomy of the order Craspedida is in need of a revision due to its controversial, paraphyletic and inconsistent systematics and nomenclature. In this study, we add molecular data (SSU and parts of the LSU rDNA) of six new Craspedida species isolated from saline, brackish and freshwater habitats to the existing knowledge. Four of these six organisms could be described as new species: Paramonosiga thecata, "Salpingoeca" euryoecia, "Salpingoeca" ventriosa, "Sphaeroeca" leprechaunica, whereas two are assigned to previous morphologically described species: "Salpingoeca" fusiformis Saville Kent, 1880 and "Salpingoeca" longipes Saville Kent, 1880. Paramonosiga is established as a new genus of the Craspedida based on its phylogenetic position. Extending the dataset by six additional sequences shows that the craspedid taxonomy is still unsolved as the type specimen Salpingoeca gracilis has not yet been sequenced and hence a clear assignment to the genus Salpingoeca is not possible. Trying to assign morphological and ecological data to phylogenetic clades is not successful. We give an improved/emended morphological diagnosis for the two redescribed species and add molecular data for all six species, shedding light on their phylogenetic position.

Stephanoeca arndti spec. nov.--first cultivation success including molecular and autecological data from a freshwater acanthoecid choanoflagellate from Samoa.

Until recently acanthoecid choanoflagellates have been described only from marine and brackish waters. Here I describe a distinct, strictly freshwater acanthoecid species from Samoa based on its morphology, ecology and molecular biological data (partial Small Subunit rDNA). The lorica of the species is characterised by two extensions at the posterior chamber which are used for attachment to the substratum. The posterior chamber is constructed of irregularly arranged costae. The anterior chamber consists of four transverse costal rings and 14-18 longitudinal costae. Despite its sturdy appearance, the lorica was extremely sensitive to water turbulence and movements of the water. The species showed a salinity tolerance of 0.5 practical salinity units with reduced growth rates and a temperature tolerance range of 20-34 °C. According to the morphology, phylogenetic analysis, and autecology of the species it was classified as a member of the genus Stephanoeca.

Acanthocorbis mongolica nov. spec.: description of the first freshwater loricate choanoflagellate (Acanthoecida) from a Mongolian lake.

A new acanthoecid choanoflagellate species, Acanthocorbis mongolica sp. nov. was found in preserved phytoplankton samples from the freshwater lake Bayan Nuur (Uvs Nuur Basin, NW Mongolia) in concentrations of up to 1.8×10(5)cellsL(-1). It is the first well-documented species of the mainly marine order Acanthoecida to be found in a freshwater lake. The lorica structures were studied with scanning electron microscopy. Key morphological features of the vase-shaped lorica are spine bases that are composed of multiple (2-4) parallel costal strips, and the existence of two transverse costae. The ecological implications of this find are discussed.

A bacterial sulfonolipid triggers multicellular development in the closest living relatives of animals.

Bacterially-produced small molecules exert profound influences on animal health, morphogenesis, and evolution through poorly understood mechanisms. In one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta, we find that rosette colony development is induced by the prey bacterium Algoriphagus machipongonensis and its close relatives in the Bacteroidetes phylum. Here we show that a rosette inducing factor (RIF-1) produced by A. machipongonensis belongs to the small class of sulfonolipids, obscure relatives of the better known sphingolipids that play important roles in signal transmission in plants, animals, and fungi. RIF-1 has extraordinary potency (femtomolar, or 10(-15) M) and S. rosetta can respond to it over a broad dynamic range-nine orders of magnitude. This study provides a prototypical example of bacterial sulfonolipids triggering eukaryotic morphogenesis and suggests molecular mechanisms through which bacteria may have contributed to the evolution of animals.DOI:http://dx.doi.org/10.7554/eLife.00013.001.

Molecular clue links bacteria to the origin of animals.

Bacteria have a role in the formation of colonies by a species of single-celled organisms whose ancestors gave rise to the animals, which suggests that bacteria might also have influenced the origin of multicellularity in animals.

Choanoflagellate lorica construction and assembly: the tectiform condition. Volkanus costatus (=Diplotheca costata).

The tectiform choanoflagellate Volkanus costatus (=Diplotheca costata) possesses a lorica comprising an outer layer of 14-18 longitudinal costae and three transverse costae. Six categories of costal strips can be distinguished on the basis of morphology. Costal strips are deposited intracellularly, upside-down, starting with the transverse costae, posterior to anterior, and then longitudinal costae, again posterior to anterior. Strips are exocytosed from the cell anteriorly and stored horizontally at the top of the collar. Cytokinesis involves inversion of the juvenile cell which is then pushed backwards into the accumulation of strips thereby restoring the normal relationship between costal strips and cell. Once free of the parent, the juvenile assembles its lorica by a forward and left-handed rotational movement. Whilst individual stages in costal strip production, storage and lorica assembly are the same in tectiform and nudiform species, nevertheless the overall procedure in tectiform species is complicated by inversion of the strip producing apparatus and cytokinesis. One explanation for this increase in complexity is that a single regulatory change has caused the coincidental inversion of costal strip production, storage and cytokinesis. The consequences of this change bequeathed tectiform cells with two important evolutionary advantages. One was the immediate inheritance by the juvenile of a lorica following division and the second was the development of transverse costae, which are mechanically superior to the helical costae of nudiform species. The outcome of these changes probably accounts for the major radiation of tectiform species within the marine environment.

Costal strip production and lorica assembly in the large tectiform choanoflagellate Diaphanoeca grandis Ellis.

Diaphanoeca grandis posseses a voluminous flask-shaped lorica comprising an outer layer of 12 longitudinal costae and an inner layer of four transverse costae. The cell is suspended just above the centre of the lorica chamber by tentacles that are attached to the anterior transverse ring. The component costal strips are superficially similar although four different strip categories can be distinguished on the basis of length and morphology. Costal strips are produced 'upside-down' within the parent cell and accumulated in a close-packed horizontal ring at the top of the inner surface of the collar. The order in which costal strips are produced is consistent, starting with those for the transverse rings, basal to anterior, and then the longitudinal costae, again with the posterior first and the anterior later. Cell division is of the classical tectiform variety with the juvenile cell being inverted and pushed backwards out of the parent lorica. Lorica assembly entails firstly the rotation of the anterior vertical strips so they become horizontal and then their movement backwards under the posterior layer of longitudinal strips. From this time onwards, lorica assembly proceeds in a standard manner with the lorica-assembling tentacles providing a forward and left-handed rotational movement.