What choanoflagellates can teach us about symbiosis.

Environmental bacteria influence many facets of choanoflagellate biology, yet surprisingly few examples of symbioses exist. We need to find out why, as choanoflagellates can help us to understand how symbiosis may have shaped the early evolution of animals.

Symbiosis: In search of a deeper understanding.

How do distinct species cofunction in symbiosis, despite conflicting interests? A new collection of articles explores emerging themes as researchers exploit modern research tools and new models to unravel how symbiotic interactions function and evolve.

Swimming, Feeding, and Inversion of Multicellular Choanoflagellate Sheets.

The recent discovery of the striking sheetlike multicellular choanoflagellate species Choanoeca flexa that dynamically interconverts between two hemispherical forms of opposite orientation raises fundamental questions in cell and evolutionary biology, as choanoflagellates are the closest living relatives of animals. It similarly motivates questions in fluid and solid mechanics concerning the differential swimming speeds in the two states and the mechanism of curvature inversion triggered by changes in the geometry of microvilli emanating from each cell. Here we develop fluid dynamical and mechanical models to address these observations and show that they capture the main features of the swimming, feeding, and inversion of C. flexa colonies, which can be viewed as active, shape-shifting polymerized membranes.

Choanoflagellates alongside diverse uncultured predatory protists consume the abundant open-ocean cyanobacterium Prochlorococcus.

Prochlorococcus is a key member of open-ocean primary producer communities. Despite its importance, little is known about the predators that consume this cyanobacterium and make its biomass available to higher trophic levels. We identify potential predators along a gradient wherein Prochlorococcus abundance increased from near detection limits (coastal California) to >200,000 cells mL-1 (subtropical North Pacific Gyre). A replicated RNA-Stable Isotope Probing experiment involving the in situ community, and labeled Prochlorococcus as prey, revealed choanoflagellates as the most active predators of Prochlorococcus, alongside a radiolarian, chrysophytes, dictyochophytes, and specific MAST lineages. These predators were not appropriately highlighted in multiyear conventional 18S rRNA gene amplicon surveys where dinoflagellates and other taxa had highest relative amplicon abundances across the gradient. In identifying direct consumers of Prochlorococcus, we reveal food-web linkages of individual protistan taxa and resolve routes of carbon transfer from the base of marine food webs.

Crystal structure of the kinase domain of a receptor tyrosine kinase from a choanoflagellate, Monosiga brevicollis.

Genomic analysis of the unicellular choanoflagellate, Monosiga brevicollis (MB), revealed the remarkable presence of cell signaling and adhesion protein domains that are characteristically associated with metazoans. Strikingly, receptor tyrosine kinases, one of the most critical elements of signal transduction and communication in metazoans, are present in choanoflagellates. We determined the crystal structure at 1.95 Å resolution of the kinase domain of the M. brevicollis receptor tyrosine kinase C8 (RTKC8, a member of the choanoflagellate receptor tyrosine kinase C family) bound to the kinase inhibitor staurospaurine. The chonanoflagellate kinase domain is closely related in sequence to mammalian tyrosine kinases (~ 40% sequence identity to the human Ephrin kinase domain EphA3) and, as expected, has the canonical protein kinase fold. The kinase is structurally most similar to human Ephrin (EphA5), even though the extracellular sensor domain is completely different from that of Ephrin. The RTKC8 kinase domain is in an active conformation, with two staurosporine molecules bound to the kinase, one at the active site and another at the peptide-substrate binding site. To our knowledge this is the first example of staurospaurine binding in the Aurora A activation segment (AAS). We also show that the RTKC8 kinase domain can phosphorylate tyrosine residues in peptides from its C-terminal tail segment which is presumably the mechanism by which it transmits the extracellular stimuli to alter cellular function.

What is it like to be a choanoflagellate? Sensation, processing and behavior in the closest unicellular relatives of animals.

All animals evolved from a single lineage of unicellular precursors more than 600 million years ago. Thus, the biological and genetic foundations for animal sensation, cognition and behavior must necessarily have arisen by modifications of pre-existing features in their unicellular ancestors. Given that the single-celled ancestors of the animal kingdom are extinct, the only way to reconstruct how these features evolved is by comparing the biology and genomic content of extant animals to their closest living relatives. Here, we reconstruct the Umwelt (the subjective, perceptive world) inhabited by choanoflagellates, a group of unicellular (or facultatively multicellular) aquatic microeukaryotes that are the closest living relatives of animals. Although behavioral research on choanoflagellates remains patchy, existing evidence shows that they are capable of chemosensation, photosensation and mechanosensation. These processes often involve specialized sensorimotor cellular appendages (cilia, microvilli, and/or filopodia) that resemble those that underlie perception in most animal sensory cells. Furthermore, comparative genomics predicts an extensive "sensory molecular toolkit" in choanoflagellates, which both provides a potential basis for known behaviors and suggests the existence of a largely undescribed behavioral complexity that presents exciting avenues for future research. Finally, we discuss how facultative multicellularity in choanoflagellates might help us understand how evolution displaced the locus of decision-making from a single cell to a collective, and how a new space of behavioral complexity might have become accessible in the process.

First putative occurrence in the fossil record of choanoflagellates, the sister group of Metazoa.

Choanoflagellates are microeukaryotes that inhabit freshwater and marine environments and have long been regarded as the closest living relatives of Metazoa. Knowledge on the evolution of choanoflagellates is key for the understanding of the ancestry of animals, and although molecular clock evidence suggests the appearance of choanoflagellates by late Neoproterozoic, no specimens of choanoflagellates are known to occur in the fossil record. Here the first putative occurrence of choanoflagellates in sediments from the Cretaceous (Cenomanian-Turonian) is described by means of several cutting-edge petrographic techniques, and a discussion of its paleoenvironmental significance is performed. Furthermore, their placement in the organic matter classification systems is argued, with a placement in the Zoomorph Subgroup (Palynomorph Group) of the dispersed organic matter classification system being proposed. Regarding the ICCP System 1994, incorporation of choanoflagellates is, at a first glance, straightforward within the liptinite group, but the definition of a new maceral may be necessary to accommodate the genetic origin of these organisms. While modern choanoflagellates may bring light to the cellular foundations of animal origins, this discovery may provide an older term of comparison to their extant specimens and provide guidelines for possible identification of these organic components in other locations and ages throughout the geological record.

Morphological and molecular reinvestigation of acanthoecid species III. - Kalathoeca cupula (Leadbeater, 1972) gen. et comb. nov. (=Stephanoeca cupula (Leadbeater) Thomsen, 1988).

Based on a further re-examination of loricate choanoflagellate species with detailed morphological (SEM/TEM) and molecular data of the SSU and LSU rRNA, the present study aims to give new insights for Stephanoeca cupula. In contrast to the original allocation within the family of tectiform reproducing species, morphological and molecular data of S. cupula sensu Leadbeater, 1972 points towards an affiliation within the nudiform reproducing family. Based on these new data, we here erect the nudiform genus Kalathoeca with its type species K. cupula gen. et comb. nov. Our data challenges morphological species assignments, as K. cupula shares its morphological lorica characteristics with tectiform reproducing species of Stephanoeca sensu stricto. Kalathoeca cupula is an interesting candidate for further investigating and understanding the evolutionary relationship of tectiform and nudiform reproducing species. Stephanoeca cupula sensu Thomsen, 1988 has been morphologically re-examined based on the renewed understanding of the morphological variability associated with S. cupula sensu Leadbeater, 1972 (=K. cupula), allowing us now to distribute the different morphological forms investigated within K. cupula and Pseudostephanoeca quasicupula.

Formation of multicellular colonies by choanoflagellates increases susceptibility to capture by amoeboid predators.

Many heterotrophic microbial eukaryotes are size-selective feeders. Some microorganisms increase their size by forming multicellular colonies. We used choanoflagellates, Salpingoeca helianthica, which can be unicellular or form multicellular colonies, to study the effects of multicellularity on vulnerability to predation by the raptorial protozoan predator, Amoeba proteus, which captures prey with pseudopodia. Videomicrography used to measure the behavior of interacting S. helianthica and A. proteus revealed that large choanoflagellate colonies were more susceptible to capture than were small colonies or single cells. Swimming colonies produced larger flow fields than did swimming unicellular choanoflagellates, and the distance of S. helianthica from A. proteus when pseudopod formation started was greater for colonies than for single cells. Prey size did not affect the number of pseudopodia formed and the time between their formation, pulsatile kinematics and speed of extension by pseudopodia, or percent of prey lost by the predator. S. helianthica did not change swimming speed or execute escape maneuvers in response to being pursued by pseudopodia, so size-selective feeding by A. proteus was due to predator behavior rather than prey escape. Our results do not support the theory that the selective advantage of becoming multicellular by choanoflagellate-like ancestors of animals was reduced susceptibility to protozoan predation.

Re-evaluating Loricate Choanoflagellate Phylogenetics: Molecular Evidence Points to the Paraphyly of Tectiform Species.

Lorica-bearing choanoflagellates belong to the order Acanthoecida, a taxon which has been consistently recovered as monophyletic in molecular phylogenies. Based upon differences in lorica development and morphology, as well as the presence or absence of a motile dispersal stage, species are labelled as either nudiform or tectiform. Whilst Acanthoecida is robustly resolved in molecular phylogenies, the placement of the root of the clade is less certain with two different positions identified in past studies. One recovered root has been placed between the nudiform family Acanthoecidae and the tectiform family Stephanoecidae. An alternative root placement falls within the tectiform species, recovering the monophyletic Acanthoecidae nested within a paraphyletic Stephanoecidae. Presented here is a 14-gene phylogeny, based upon nucleotide and amino acid sequences, which strongly supports tectiform paraphyly. The horizontal transfer of a ribosomal protein gene, from a possible SAR donor, into a subset of acanthoecid species provides further, independent, support for this root placement. Differing patterns of codon usage bias across the choanoflagellates are proposed as the cause of artefactual phylogenetic signals that lead to the recovery of tectiform monophyly.

Three-dimensional flagella structures from animals' closest unicellular relatives, the Choanoflagellates.

In most eukaryotic organisms, cilia and flagella perform a variety of life-sustaining roles related to environmental sensing and motility. Cryo-electron microscopy has provided considerable insight into the morphology and function of flagellar structures, but studies have been limited to less than a dozen of the millions of known eukaryotic species. Ultrastructural information is particularly lacking for unicellular organisms in the Opisthokonta clade, leaving a sizeable gap in our understanding of flagella evolution between unicellular species and multicellular metazoans (animals). Choanoflagellates are important aquatic heterotrophs, uniquely positioned within the opisthokonts as the metazoans' closest living unicellular relatives. We performed cryo-focused ion beam milling and cryo-electron tomography on flagella from the choanoflagellate species Salpingoeca rosetta. We show that the axonemal dyneins, radial spokes, and central pair complex in S. rosetta more closely resemble metazoan structures than those of unicellular organisms from other suprakingdoms. In addition, we describe unique features of S. rosetta flagella, including microtubule holes, microtubule inner proteins, and the flagellar vane: a fine, net-like extension that has been notoriously difficult to visualize using other methods. Furthermore, we report barb-like structures of unknown function on the extracellular surface of the flagellar membrane. Together, our findings provide new insights into choanoflagellate biology and flagella evolution between unicellular and multicellular opisthokonts.

Significance of the Nudiform and Tectiform Modes of Silica Deposition, Lorica Assembly and Cell Division in Choanoflagellates as Exemplified by Stephanoeca diplocostata and Enibas spp.

The deposition of silicified costal strips and lorica assembly in choanoflagellates is precisely linked to the cell cycle. A minority of species undergo nudiform division whereby a loricate cell divides to produce a naked daughter cell that deposits a set of costal strips and then assembles a lorica. Most species undergo tectiform division whereby a parent loricate cell produces a set of costal strips, divides and passes on the stored strips to a daughter cell that immediately assembles a lorica. Many phylogenetic analyses recover nudiform and tectiform species as sister-clades giving the impression that they are distinct evolutionary lineages. However, the tectiform species Stephanoeca diplocostata is capable of undergoing nudiform division and depositing costal strips and assembling a lorica with certain modifications in a nudiform manner. The recent discovery of a new genus, Enibas, comprising species with Stephanoeca-like loricae that undergo nudiform cell division and on phylogenetic analysis occur as a sister group to other nudiform species has drawn attention to whether there are also unique features in lorica construction. A range of Enibas loricae is illustrated and it appears that there are unique features which might be interpreted as being derived from a Stephanoeca-like ancestor.

Morphological and molecular reinvestigation of acanthoecid species II. - Pseudostephanoeca paucicostata (Tong et al., 1998) gen. et comb. nov. (= Stephanoeca diplocostata var. paucicostata Throndsen, 1969) including also the description of Pseudostephanoeca quasicupula sp. nov. and Stephanoeca ellisfiordensis sp. nov.

The present study has revealed morphological differences of species assigned to the acanthoecid choanoflagellate genus Stephanoeca based on the relative position of transverse and longitudinal costae in the anterior lorica chamber. A detailed re-examination of published material of S. paucicostata Tong et al., 1998 combined with new data has shown that the species characteristic, a double transverse costa, is clearly located outside of the longitudinal costae excluding this taxon from an affiliation to Stephanoeca sensu stricto, confirmed also by molecular data of a clonal culture from Kos Islands, Greece. Two specimens of S. paucicostata with only a single, interiorly positioned transverse costa, namely from Marchant et al. (1987) and Nitsche et al. (2011) were erroneously assigned to this species. These facts have here led to the establishment of a new genus, Pseudostephanoeca with its type species P. paucicostata, and the description of S. ellisfiordensis for the two specimens mentioned above, corroborated by new material from Antarctica. In addition, two specimens previously assigned to S. cupula have been shown to share the core characteristic of Pseudostephanoeca with exteriorly positioned transverse elements. This has led to the description of the new species P. quasicupula including also new material from Iceland.

Flow cytometric sorting of loricate choanoflagellates from the oligotrophic ocean.

It is challenging to study protists with extensive, loosely-associated extracellular structures because of the problems with keeping specimens intact. Here we have tested the suitability of high-speed flow cytometric sorting as a tool for studying such protists using oceanic loricate choanoflagellates as a model. We chose choanoflagellates because their lorica-to-cell volume ratio is > 10 and the voluminous loricae, i.e., the siliceous cell baskets essential for taxonomic identification, only loosely enclose the cells. Besides, owing to low concentrations, choanoflagellates are grossly under-sampled in the oligotrophic ocean. On four research cruises the small heterotrophic protists from samples collected in the photic layer of the South Atlantic and South Pacific oligotrophic (sub)tropical gyres and adjacent mesotrophic waters were flow sorted at sea for electron microscopy ashore. Among the flow-sorted protozoa we were able to select loricate choanoflagellates to assess their species diversity and concentrations. The well-preserved loricae of flow-sorted choanoflagellates made identification of 29 species from 14 genera possible. In the oligotrophic waters, we found neither endemic species nor evident morphological adaptations other than a tendency for lighter silicification of loricae. Common sightings of specimens storing extra costae in preparation for division, indicate choanoflagellates thriving in oligotrophic waters rather than enduring them. Thus, this case study demonstrates that high-speed flow sorting can assist in studying protists with extracellular structures 16-78× bigger than the enclosed cell.

The choanoflagellate pore-forming lectin SaroL-1 punches holes in cancer cells by targeting the tumor-related glycosphingolipid Gb3.

Choanoflagellates are primitive protozoa used as models for animal evolution. They express a large variety of multi-domain proteins contributing to adhesion and cell communication, thereby providing a rich repertoire of molecules for biotechnology. Adhesion often involves proteins adopting a ß-trefoil fold with carbohydrate-binding properties therefore classified as lectins. Sequence database screening with a dedicated method resulted in TrefLec, a database of 44714 ß-trefoil candidate lectins across 4497 species. TrefLec was searched for original domain combinations, which led to single out SaroL-1 in the choanoflagellate Salpingoeca rosetta, that contains both ß-trefoil and aerolysin-like pore-forming domains. Recombinant SaroL-1 is shown to bind galactose and derivatives, with a stronger affinity for cancer-related α-galactosylated epitopes such as the glycosphingolipid Gb3, when embedded in giant unilamellar vesicles or cell membranes. Crystal structures of complexes with Gb3 trisaccharide and GalNAc provided the basis for building a model of the oligomeric pore. Finally, recognition of the αGal epitope on glycolipids required for hemolysis of rabbit erythrocytes suggests that toxicity on cancer cells is achieved through carbohydrate-dependent pore-formation.

The microbiome of a bacterivorous marine choanoflagellate contains a resource-demanding obligate bacterial associate.

Microbial predators such as choanoflagellates are key players in ocean food webs. Choanoflagellates, which are the closest unicellular relatives of animals, consume bacteria and also exhibit marked biological transitions triggered by bacterial compounds, yet their native microbiomes remain uncharacterized. Here we report the discovery of a ubiquitous, uncultured bacterial lineage we name Candidatus Comchoanobacterales ord. nov., related to the human pathogen Coxiella and physically associated with the uncultured marine choanoflagellate Bicosta minor. We analyse complete 'Comchoano' genomes acquired after sorting single Bicosta cells, finding signatures of obligate host-dependence, including reduction of pathways encoding glycolysis, membrane components, amino acids and B-vitamins. Comchoano encode the necessary apparatus to import energy and other compounds from the host, proteins for host-cell associations and a type IV secretion system closest to Coxiella's that is expressed in Pacific Ocean metatranscriptomes. Interactions between choanoflagellates and their microbiota could reshape the direction of energy and resource flow attributed to microbial predators, adding complexity and nuance to marine food webs.

Total Synthesis and Functional Evaluation of IORs, Sulfonolipid-based Inhibitors of Cell Differentiation in Salpingoeca rosetta.

The choanoflagellate Salpingoeca rosetta is an important model system to study the evolution of multicellularity. In this study we developed a new, modular, and scalable synthesis of sulfonolipid IOR-1A (six steps, 27 % overall yield), which acts as bacterial inhibitor of rosette formation in S. rosetta. The synthesis features a decarboxylative cross-coupling reaction of a sulfonic acid-containing tartaric acid derivative with alkyl zinc reagents. Synthesis of 15 modified IOR-1A derivatives, including fluorescent and photoaffinity-based probes, allowed quantification of IOR-1A, localization studies within S. rosetta cells, and evaluation of structure-activity relations. In a proof of concept study, an inhibitory bifunctional probe was employed in proteomic profiling studies, which allowed to deduce binding partners in bacteria and S. rosetta. These results showcase the power of synthetic chemistry to decipher the biochemical basis of cell differentiation processes within S. rosetta.

Evolution: NO signaling at the stem of animal life.

Nitric oxide (NO), an ancient gaseous signaling molecule, regulates several physiological processes across the kingdoms. A new study describes how NO controls collective cell contractions in the closest animal relatives, the choanoflagellates, to switch from feeding to swimming away.

Nitric oxide signaling controls collective contractions in a colonial choanoflagellate.

Although signaling by the gaseous molecule nitric oxide (NO) regulates key physiological processes in animals, including contractility,1-3 immunity,4,5 development,6-9 and locomotion,10,11 the early evolution of animal NO signaling remains unclear. To reconstruct the role of NO in the animal stem lineage, we set out to study NO signaling in choanoflagellates, the closest living relatives of animals.12 In animals, NO produced by the nitric oxide synthase (NOS) canonically signals through cGMP by activating soluble guanylate cyclases (sGCs).13,14 We surveyed the distribution of the NO signaling pathway components across the diversity of choanoflagellates and found three species that express NOS (of either bacterial or eukaryotic origin), sGCs, and downstream genes previously shown to be involved in the NO/cGMP pathway. One of the species coexpressing sGCs and a bacterial-type NOS, Choanoeca flexa, forms multicellular sheets that undergo collective contractions controlled by cGMP.15 We found that treatment with NO induces cGMP synthesis and contraction in C. flexa. Biochemical assays show that NO directly binds C. flexa sGC1 and stimulates its cyclase activity. The NO/cGMP pathway acts independently from other inducers of C. flexa contraction, including mechanical stimuli and heat, but sGC activity is required for contractions induced by light-to-dark transitions. The output of NO signaling in C. flexa-contractions resulting in a switch from feeding to swimming-resembles the effect of NO in sponges1-3 and cnidarians,11,16,17 where it interrupts feeding and activates contractility. These data provide insights into the biology of the first animals and the evolution of NO signaling.

Premetazoan Origin of Neuropeptide Signaling.

Neuropeptides are a diverse class of signaling molecules in metazoans. They occur in all animals with a nervous system and also in neuron-less placozoans. However, their origin has remained unclear because no neuropeptide shows deep homology across lineages, and none have been found in sponges. Here, we identify two neuropeptide precursors, phoenixin (PNX) and nesfatin, with broad evolutionary conservation. By database searches, sequence alignments, and gene-structure comparisons, we show that both precursors are present in bilaterians, cnidarians, ctenophores, and sponges. We also found PNX and a secreted nesfatin precursor homolog in the choanoflagellate Salpingoeca rosetta. PNX, in particular, is highly conserved, including its cleavage sites, suggesting that prohormone processing occurs also in choanoflagellates. In addition, based on phyletic patterns and negative pharmacological assays, we question the originally proposed GPR-173 (SREB3) as a PNX receptor. Our findings revealed that secreted neuropeptide homologs derived from longer precursors have premetazoan origins and thus evolved before neurons.