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.

Choanoflagellates and the ancestry of neurosecretory vesicles.

Neurosecretory vesicles are highly specialized trafficking organelles that store neurotransmitters that are released at presynaptic nerve endings and are, therefore, important for animal cell-cell signalling. Despite considerable anatomical and functional diversity of neurons in animals, the protein composition of neurosecretory vesicles in bilaterians appears to be similar. This similarity points towards a common evolutionary origin. Moreover, many putative homologues of key neurosecretory vesicle proteins predate the origin of the first neurons, and some even the origin of the first animals. However, little is known about the molecular toolkit of these vesicles in non-bilaterian animals and their closest unicellular relatives, making inferences about the evolutionary origin of neurosecretory vesicles extremely difficult. By comparing 28 proteins of the core neurosecretory vesicle proteome in 13 different species, we demonstrate that most of the proteins are present in unicellular organisms. Surprisingly, we find that the vesicular membrane-associated soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein synaptobrevin is localized to the vesicle-rich apical and basal pole in the choanoflagellate Salpingoeca rosetta. Our 3D vesicle reconstructions reveal that the choanoflagellates S. rosetta and Monosiga brevicollis exhibit a polarized and diverse vesicular landscape reminiscent of the polarized organization of chemical synapses that secrete the content of neurosecretory vesicles into the synaptic cleft. This study sheds light on the ancestral molecular machinery of neurosecretory vesicles and provides a framework to understand the origin and evolution of secretory cells, synapses and neurons. This article is part of the theme issue 'Basal cognition: multicellularity, neurons and the cognitive lens'.

Selective factors in the evolution of multicellularity in choanoflagellates.

Choanoflagellates, unicellular eukaryotes that can form multicellular colonies by cell division and that share a common ancestor with animals, are used as a model system to study functional consequences of being unicellular versus colonial. This review examines performance differences between unicellular and multicellular choanoflagellates in swimming, feeding, and avoiding predation, to provide insights about possible selective advantages of being multicellular for the protozoan ancestors of animals. Each choanoflagellate cell propels water by beating a single flagellum and captures bacterial prey on a collar of microvilli around the flagellum. Formation of multicellular colonies does not improve the swimming performance, but the flux of prey-bearing water to the collars of some of the cells in colonies of certain configurations can be greater than for single cells. Colony geometry appears to affect whether cells in colonies catch more prey per cell per time than do unicellular choanoflagellates. Although multicellular choanoflagellates show chemokinetic behavior in response to oxygen, only the unicellular dispersal stage (fast swimmers without collars) use pH signals to aggregate in locations where bacterial prey might be abundant. Colonies produce larger hydrodynamic signals than do single cells, and raptorial protozoan predators capture colonies while ignoring single cells. In contrast, ciliate predators entrain both single cells and colonies in their feeding currents, but reject larger colonies, whereas passive heliozoan predators show no preference. Thus, the ability of choanoflagellate cells to differentiate into different morphotypes, including multicellular forms, in response to variable aquatic environments might have provided a selective advantage to the ancestors of animals.

Loricate choanoflagellates (Acanthoecida) from warm water seas. VI. Pleurasiga Schiller and Parvicorbicula Deflandre.

The loricate choanoflagellate genera Pleurasiga and Parvicorbicula are taxonomically ambiguous. Pleurasiga because of the uncertainty that relates to the true identity of the type species, and Parvicorbicula because too many newly described species over time have been dumped here in lack of better options. While all species currently allocated to the genus Pleurasiga (with the exception of the type species) are observed in our samples from the global warm water belt, the genus Parvicorbicula is represented by just a few and mostly infrequently recorded taxa. Two new species, viz. Pl. quadrangiella sp. nov. and Pl. minutissima sp. nov., are described here. While the former is closely related to Pl. echinocostata, the latter is reminiscent of Pl. minima. Core species of Pleurasiga and Parvicorbicula deviate from the vast majority of loricate choanoflagellates in having both the anterior and the mid-lorica transverse costae located exterior to the longitudinal costae. In Pl. quadrangiella there is no mid-lorica transverse costa but rather a small posterior transverse costa located inside the longitudinal costae. In Pl. minutissima the mid-lorica transverse costa has extensive costal strip overlaps which reveal patterns of costal strip junctions that deviate from the norm.

Synergistic Cues from Diverse Bacteria Enhance Multicellular Development in a Choanoflagellate.

Bacteria regulate the life histories of diverse eukaryotes, but relatively little is known about how eukaryotes interpret and respond to multiple bacterial cues encountered simultaneously. To explore how a eukaryote might respond to a combination of bioactive molecules from multiple bacteria, we treated the choanoflagellate Salpingoeca rosetta with two sets of bacterial cues, one that induces mating and another that induces multicellular development. We found that simultaneous exposure to both sets of cues enhanced multicellular development in S. rosetta, eliciting both larger multicellular colonies and an increase in the number of colonies. Thus, rather than conveying conflicting sets of information, these distinct bacterial cues synergize to augment multicellular development. This study demonstrates how a eukaryote can integrate and modulate its response to cues from diverse bacteria, underscoring the potential impact of complex microbial communities on eukaryotic life histories.IMPORTANCE Eukaryotic biology is profoundly influenced by interactions with diverse environmental and host-associated bacteria. However, it is not well understood how eukaryotes interpret multiple bacterial cues encountered simultaneously. This question has been challenging to address because of the complexity of many eukaryotic model systems and their associated bacterial communities. Here, we studied a close relative of animals, the choanoflagellate Salpingoeca rosetta, to explore how eukaryotes respond to diverse bacterial cues. We found that a bacterial chondroitinase that induces mating on its own can also synergize with bacterial lipids that induce multicellular "rosette" development. When encountered together, these cues enhance rosette development, resulting in both the formation of larger rosettes and an increase in the number of rosettes compared to rosette development in the absence of the chondroitinase. These findings highlight how synergistic interactions among bacterial cues can influence the biology of eukaryotes.

Rosette Colonies of Choanoflagellates (Salpingoeca rosetta) Show Increased Food Vacuole Formation Compared with Single Swimming Cells.

Choanoflagellates exist as both single-celled and colonial forms and filter-feed by generating water currents using a single apical flagellum. Hydrodynamic modeling studies have differed in predictions of whether single cells or colonies produce greater fluid flow to enhance feeding, and a recent study reported no increase in feeding efficiency of stalked colonies of choanoflagellates compared with single cells. We report that rosette colonies of Salpingoeca rosetta demonstrate higher rates of food vacuole formation compared with unicellular, slow swimmers.

Growth and single cell kinetics of the loricate choanoflagellate Diaphanoeca grandis.

Choanoflagellates are common members of planktonic communities. Some have complex life histories that involve transitions between multiple cell stages. We have grown the loricate choanoflagellate Diaphanoeca grandis on the bacterium Pantoea sp. and integrated kinetic observations at the culture level and at the single cell level. The life history of D. grandis includes a cell division cycle with a number of recognisable cell stages. Mature, loricate D. grandis were immobile and settled on the bottom substratum. Daughter cells were ejected from the lorica 30 min. after cell division, became motile and glided on the bottom substratum until they assembled a lorica. Single cell kinetics could explain overall growth kinetics in D. grandis cultures. The specific growth rate was 0.72 day-1 during exponential growth while mature D. grandis produced daughter cells at a rate of 0.9 day-1. Daughter cells took about 1.2 h to mature. D. grandis was able to abandon and replace its lorica, an event that delayed daughter cell formation by more than 2 days. The frequency of daughter cell formation varied considerably among individuals and single cell kinetics demonstrated an extensive degree of heterogeneity in D. grandis cultures, also when growth appeared to be balanced.

Light-regulated collective contractility in a multicellular choanoflagellate.

Collective cell contractions that generate global tissue deformations are a signature feature of animal movement and morphogenesis. However, the origin of collective contractility in animals remains unclear. While surveying the Caribbean island of Curaçao for choanoflagellates, the closest living relatives of animals, we isolated a previously undescribed species (here named Choanoeca flexa sp. nov.) that forms multicellular cup-shaped colonies. The colonies rapidly invert their curvature in response to changing light levels, which they detect through a rhodopsin-cyclic guanosine monophosphate pathway. Inversion requires actomyosin-mediated apical contractility and allows alternation between feeding and swimming behavior. C. flexa thus rapidly converts sensory inputs directly into multicellular contractions. These findings may inform reconstructions of hypothesized animal ancestors that existed before the evolution of specialized sensory and contractile cells.

Loricate choanoflagellates (Acanthoecida) from warm water seas. V. Thomsenella Özdikmen (= Platypleura Thomsen).

Loricate choanoflagellate genera that incorporate flattened costal strips in the lorica (i.e. Calotheca, Stephanacantha, Thomsenella (= Platypleura) and Syndetophyllum) are prevalent in warm water habitats. The genus Thomsenella (=Platypleura) thus comprises four species and three of these have an Andaman Sea type locality. Our ongoing examination of loricate choanoflagellate material from all major warm water oceans has provided us with the opportunity of revisiting species of Thomsenella in order to test and fortunately verify, in a morpho-specific context, the robustness of the species matrix initially defined.

Filter-feeding in Colonial Protists.

Many protists form cell colonies. Among them several are filter-feeders depending on suspended food particles such as bacteria. It has been suggested that the formation of colonies enhances feeding efficiency and implied that - in the case of colonial choanoflagellates - it was an adaptive trait that led to the evolution of metazoans. Here it is shown experimentally - for a colonial peritrich ciliate and for a choanoflagellate - that colony-formation does not enhance the efficiency of filter-feeding relative to solitary cells and that the adaptive significance of cell colony-formation must have some other explanation.

The architecture of cell differentiation in choanoflagellates and sponge choanocytes.

Although collar cells are conserved across animals and their closest relatives, the choanoflagellates, little is known about their ancestry, their subcellular architecture, or how they differentiate. The choanoflagellate Salpingoeca rosetta expresses genes necessary for animal development and can alternate between unicellular and multicellular states, making it a powerful model for investigating the origin of animal multicellularity and mechanisms underlying cell differentiation. To compare the subcellular architecture of solitary collar cells in S. rosetta with that of multicellular 'rosette' colonies and collar cells in sponges, we reconstructed entire cells in 3D through transmission electron microscopy on serial ultrathin sections. Structural analysis of our 3D reconstructions revealed important differences between single and colonial choanoflagellate cells, with colonial cells exhibiting a more amoeboid morphology consistent with higher levels of macropinocytotic activity. Comparison of multiple reconstructed rosette colonies highlighted the variable nature of cell sizes, cell-cell contact networks, and colony arrangement. Importantly, we uncovered the presence of elongated cells in some rosette colonies that likely represent a distinct and differentiated cell type, pointing toward spatial cell differentiation. Intercellular bridges within choanoflagellate colonies displayed a variety of morphologies and connected some but not all neighbouring cells. Reconstruction of sponge choanocytes revealed ultrastructural commonalities but also differences in major organelle composition in comparison to choanoflagellates. Together, our comparative reconstructions uncover the architecture of cell differentiation in choanoflagellates and sponge choanocytes and constitute an important step in reconstructing the cell biology of the last common ancestor of animals.

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.

Effects of cell morphology and attachment to a surface on the hydrodynamic performance of unicellular choanoflagellates.

Choanoflagellates, eukaryotes that are important predators on bacteria in aquatic ecosystems, are closely related to animals and are used as a model system to study the evolution of animals from protozoan ancestors. The choanoflagellate Salpingoeca rosetta has a complex life cycle with different morphotypes, some unicellular and some multicellular. Here we use computational fluid dynamics to study the hydrodynamics of swimming and feeding by different unicellular stages of S. rosetta: a swimming cell with a collar of prey-capturing microvilli surrounding a single flagellum, a thecate cell attached to a surface and a dispersal-stage cell with a slender body, long flagellum and short collar. We show that a longer flagellum increases swimming speed, longer microvilli reduce speed and cell shape only affects speed when the collar is very short. The flux of prey-carrying water into the collar capture zone is greater for swimming than sessile cells, but this advantage decreases with collar size. Stalk length has little effect on flux for sessile cells. We show that ignoring the collar, as earlier models have done, overestimates flux and greatly overestimates the benefit to feeding performance of swimming versus being attached, and of a longer stalk for attached cells.

Transfection of choanoflagellates illuminates their cell biology and the ancestry of animal septins.

As the closest living relatives of animals, choanoflagellates offer unique insights into animal origins and core mechanisms underlying animal cell biology. However, unlike traditional model organisms, such as yeast, flies, and worms, choanoflagellates have been refractory to DNA delivery methods for expressing foreign genes. Here we report a robust method for expressing transgenes in the choanoflagellate Salpingoeca rosetta, overcoming barriers that have previously hampered DNA delivery and expression. To demonstrate how this method accelerates the study of S. rosetta cell biology, we engineered a panel of fluorescent protein markers that illuminate key features of choanoflagellate cells. We then investigated the localization of choanoflagellate septins, a family of GTP-binding cytoskeletal proteins that are hypothesized to regulate multicellular rosette development in S. rosetta. Fluorescently tagged septins localized to the basal poles of S. rosetta single cells and rosettes in a pattern resembling septin localization in animal epithelia. The establishment of transfection in S. rosetta and its application to the study of septins represent critical advances in the use of S. rosetta as an experimental model for investigating choanoflagellate cell biology, core mechanisms underlying animal cell biology, and the origin of animals.

Mating in the Closest Living Relatives of Animals Is Induced by a Bacterial Chondroitinase.

We serendipitously discovered that the marine bacterium Vibrio fischeri induces sexual reproduction in one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta. Although bacteria influence everything from nutrition and metabolism to cell biology and development in eukaryotes, bacterial regulation of eukaryotic mating was unexpected. Here, we show that a single V. fischeri protein, the previously uncharacterized EroS, fully recapitulates the aphrodisiac-like activity of live V. fischeri. EroS is a chondroitin lyase; although its substrate, chondroitin sulfate, was previously thought to be an animal synapomorphy, we demonstrate that S. rosetta produces chondroitin sulfate and thus extend the ancestry of this important glycosaminoglycan to the premetazoan era. Finally, we show that V. fischeri, purified EroS, and other bacterial chondroitin lyases induce S. rosetta mating at environmentally relevant concentrations, suggesting that bacteria likely regulate choanoflagellate mating in nature.

Origin of animal multicellularity: precursors, causes, consequences-the choanoflagellate/sponge transition, neurogenesis and the Cambrian explosion.

Evolving multicellularity is easy, especially in phototrophs and osmotrophs whose multicells feed like unicells. Evolving animals was much harder and unique; probably only one pathway via benthic 'zoophytes' with pelagic ciliated larvae allowed trophic continuity from phagocytic protozoa to gut-endowed animals. Choanoflagellate protozoa produced sponges. Converting sponge flask cells mediating larval settling to synaptically controlled nematocysts arguably made Cnidaria. I replace Haeckel's gastraea theory by a sponge/coelenterate/bilaterian pathway: Placozoa, hydrozoan diploblasty and ctenophores were secondary; stem anthozoan developmental mutations arguably independently generated coelomate bilateria and ctenophores. I emphasize animal origin's conceptual aspects (selective, developmental) related to feeding modes, cell structure, phylogeny of related protozoa, sequence evidence, ecology and palaeontology. Epithelia and connective tissue could evolve only by compensating for dramatically lower feeding efficiency that differentiation into non-choanocytes entails. Consequentially, larger bodies enabled filtering more water for bacterial food and harbouring photosynthetic bacteria, together adding more food than cell differentiation sacrificed. A hypothetical presponge of sessile triploblastic sheets (connective tissue sandwiched between two choanocyte epithelia) evolved oogamy through selection for larger dispersive ciliated larvae to accelerate benthic trophic competence and overgrowing protozoan competitors. Extinct Vendozoa might be elaborations of this organismal grade with choanocyte-bearing epithelia, before poriferan water channels and cnidarian gut/nematocysts/synapses evolved.This article is part of the themed issue 'Evo-devo in the genomics era, and the origins of morphological diversity'.

Filter-feeding, near-field flows, and the morphologies of colonial choanoflagellates.

Efficient uptake of prey and nutrients from the environment is an important component in the fitness of all microorganisms, and its dependence on size may reveal clues to the origins of evolutionary transitions to multicellularity. Because potential benefits in uptake rates must be viewed in the context of other costs and benefits of size, such as varying predation rates and the increased metabolic costs associated with larger and more complex body plans, the uptake rate itself is not necessarily that which is optimized by evolution. Uptake rates can be strongly dependent on local organism geometry and its swimming speed, providing selective pressure for particular arrangements. Here we examine these issues for choanoflagellates, filter-feeding microorganisms that are the closest relatives of the animals. We explore the different morphological variations of the choanoflagellate Salpingoeca rosetta, which can exist as a swimming cell, as a sessile thecate cell, and as colonies of cells in various shapes. In the absence of other requirements and in a homogeneously nutritious environment, we find that the optimal strategy to maximize filter-feeding by the collar of microvilli is to swim fast, which favors swimming unicells. In large external flows, the sessile thecate cell becomes advantageous. Effects of prey diffusion are discussed and also found to be to the advantage of the swimming unicell.

Aerotaxis in the closest relatives of animals.

As the closest unicellular relatives of animals, choanoflagellates serve as useful model organisms for understanding the evolution of animal multicellularity. An important factor in animal evolution was the increasing ocean oxygen levels in the Precambrian, which are thought to have influenced the emergence of complex multicellular life. As a first step in addressing these conditions, we study here the response of the colony-forming choanoflagellate Salpingoeca rosetta to oxygen gradients. Using a microfluidic device that allows spatio-temporal variations in oxygen concentrations, we report the discovery that S. rosetta displays positive aerotaxis. Analysis of the spatial population distributions provides evidence for logarithmic sensing of oxygen, which enhances sensing in low oxygen neighborhoods. Analysis of search strategy models on the experimental colony trajectories finds that choanoflagellate aerotaxis is consistent with stochastic navigation, the statistics of which are captured using an effective continuous version based on classical run-and-tumble chemotaxis.

Seasonal Occurrence of Loricate Choanoflagellates in Danish Inner Waters.

It is a trend in loricate choanoflagellate research that our knowledge of species diversity is insufficient in terms of understanding annual successional changes at any specific locality, whereas there is a fairly decent coverage worldwide - at least in more coastal realms - in terms of biodiversity within more narrowly defined time windows. To help address this knowledge gap, we have compiled all available loricate choanoflagellate occurrence data from Danish sampling sites covering an overall time span of close to four decades. The close to 100 samples analysed have a good annual coverage and they encompass in total more than 50 species. We demonstrate clear successional trends among well-defined clusters of species. A large contingent of 'non-native' species, which are in a global context largely considered part of the loricate choanoflagellate warm water community, occurred in September 2014 samples from the Baltic Sea entrance, i.e. the Sound between Denmark and Sweden. While the occurrence of these species is likely due to a large inflow of southern Atlantic water, we also discuss whether the findings may instead reflect recent and more permanent climate change-induced alterations to choanoflagellate biodiversity in inner Danish waters.

Bacterial lipids activate, synergize, and inhibit a developmental switch in choanoflagellates.

In choanoflagellates, the closest living relatives of animals, multicellular rosette development is regulated by environmental bacteria. The simplicity of this evolutionarily relevant interaction provides an opportunity to identify the molecules and regulatory logic underpinning bacterial regulation of development. We find that the rosette-inducing bacterium Algoriphagus machipongonensis produces three structurally divergent classes of bioactive lipids that, together, activate, enhance, and inhibit rosette development in the choanoflagellate Salpingoeca rosetta. One class of molecules, the lysophosphatidylethanolamines (LPEs), elicits no response on its own but synergizes with activating sulfonolipid rosette-inducing factors (RIFs) to recapitulate the full bioactivity of live Algoriphagus. LPEs, although ubiquitous in bacteria and eukaryotes, have not previously been implicated in the regulation of a host-microbe interaction. This study reveals that multiple bacterially produced lipids converge to activate, enhance, and inhibit multicellular development in a choanoflagellate.