Divergent Ca2+/calmodulin feedback regulation of CaV1 and CaV2 voltage-gated calcium channels evolved in the common ancestor of Placozoa and Bilateria.

CaV1 and CaV2 voltage-gated calcium channels evolved from an ancestral CaV1/2 channel via gene duplication somewhere near the stem animal lineage. The divergence of these channel types led to distinguishing functional properties that are conserved among vertebrates and bilaterian invertebrates and contribute to their unique cellular roles. One key difference pertains to their regulation by calmodulin (CaM), wherein bilaterian CaV1 channels are uniquely subject to pronounced, buffer-resistant Ca2+/CaM-dependent inactivation, permitting negative feedback regulation of calcium influx in response to local cytoplasmic Ca2+ rises. Early diverging, nonbilaterian invertebrates also possess CaV1 and CaV2 channels, but it is unclear whether they share these conserved functional features. The most divergent animals to possess both CaV1 and CaV2 channels are placozoans such as Trichoplax adhaerens, which separated from other animals over 600 million years ago shortly after their emergence. Hence, placozoans can provide important insights into the early evolution of CaV1 and CaV2 channels. Here, we build upon previous characterization of Trichoplax CaV channels by determining the cellular expression and ion-conducting properties of the CaV1 channel orthologue, TCaV1. We show that TCaV1 is expressed in neuroendocrine-like gland cells and contractile dorsal epithelial cells. In vitro, this channel conducts dihydropyridine-insensitive, high-voltage-activated Ca2+ currents with kinetics resembling those of rat CaV1.2 but with left-shifted voltage sensitivity for activation and inactivation. Interestingly, TCaV1, but not TCaV2, exhibits buffer-resistant Ca2+/CaM-dependent inactivation, indicating that this functional divergence evolved prior to the emergence of bilaterian animals and may have contributed to their unique adaptation for cytoplasmic Ca2+ signaling within various cellular contexts.

Chemical cognition: chemoconnectomics and convergent evolution of integrative systems in animals.

Neurons underpin cognition in animals. However, the roots of animal cognition are elusive from both mechanistic and evolutionary standpoints. Two conceptual frameworks both highlight and promise to address these challenges. First, we discuss evidence that animal neural and other integrative systems evolved more than once (convergent evolution) within basal metazoan lineages, giving us unique experiments by Nature for future studies. The most remarkable examples are neural systems in ctenophores and neuroid-like systems in placozoans and sponges. Second, in addition to classical synaptic wiring, a chemical connectome mediated by hundreds of signal molecules operates in tandem with neurons and is the most information-rich source of emerging properties and adaptability. The major gap-dynamic, multifunctional chemical micro-environments in nervous systems-is not understood well. Thus, novel tools and information are needed to establish mechanistic links between orchestrated, yet cell-specific, volume transmission and behaviors. Uniting what we call chemoconnectomics and analyses of the cellular bases of behavior in basal metazoan lineages arguably would form the foundation for deciphering the origins and early evolution of elementary cognition and intelligence.

The enigmatic Placozoa part 2: Exploring evolutionary controversies and promising questions on earth and in space.

The placozoan Trichoplax adhaerens has been bridging gaps between research disciplines like no other animal. As outlined in part 1, placozoans have been subject of hot evolutionary debates and placozoans have challenged some fundamental evolutionary concepts. Here in part 2 we discuss the exceptional genetics of the phylum Placozoa and point out some challenging model system applications for the best known species, Trichoplax adhaerens.

The enigmatic Placozoa part 1: Exploring evolutionary controversies and poor ecological knowledge.

The placozoan Trichoplax adhaerens is a tiny hairy plate and more simply organized than any other living metazoan. After its original description by F.E. Schulze in 1883, it attracted attention as a potential model for the ancestral state of metazoan organization, the "Urmetazoon". Trichoplax lacks any kind of symmetry, organs, nerve cells, muscle cells, basal lamina, and extracellular matrix. Furthermore, the placozoan genome is the smallest (not secondarily reduced) genome of all metazoan genomes. It harbors a remarkably rich diversity of genes and has been considered the best living surrogate for a metazoan ancestor genome. The phylum Placozoa presently harbors three formally described species, while several dozen "cryptic" species are yet awaiting their description. The phylogenetic position of placozoans has recently become a contested arena for modern phylogenetic analyses and view-driven claims. Trichoplax offers unique prospects for understanding the minimal requirements of metazoan animal organization and their corresponding malfunctions.

[Spatial Models of Piezo Proteins and Protein-Protein Interaction Networks in Trichoplax Animals (Placozoa)].

The marine free-living organism Trichoplax (phylum Placozoa) resembles a unicellular amoeba in shape and type of movement. Trichoplax diverged from the main evolutionary tree in the Neoproterozoic Era. Trichoplax provides one of the simplest models of multicellular animals and a strong example of how cells of an organism interact to form an ensemble during its development and movement. Two orthologs of the mouse Piezo1 protein (6B3R) were found in two Trichoplax haplotypes, H1 and H2, as a result of a search for similar sequences in the NCBI databases. Spatial models of the respective proteins XP_002112008.1 and RDD46920.1 were created via a structural alignment with 6KG7 (mouse Piezo2) template. Their domain structures were analyzed, and a limited graph of protein-protein interactions was constructed for the hypothetical mechanosensor XP_002112008.1. The possibility of signal transduction from the mechanoreceptor to membrane complexes, the cytoplasm, and the cell nucleus was shown. Trichoplax mechanoreceptors were assumed to play a role in perception of force stimuli from neighbor cells and the environment. Based on the results, the primitive Trichoplax organism was proposed as the simplest multicellular model of mechanical and morphogenetic movements.

Observing Phylum-Level Metazoan Diversity by Environmental DNA Analysis at the Ushimado Area in the Seto Inland Sea.

The dynamics of microscopic marine plankton in coastal areas is a fundamental theme in marine biodiversity research, but studies have been limited because the only available methodology was collection of plankton using plankton-nets and microscopic observation. In recent years, environmental DNA (eDNA) analysis has exhibited potential for conducting comprehensive surveys of marine plankton diversity in water at fixed points and depths in the ocean. However, few studies have examined how eDNA analysis reflects the actual distribution and dynamics of organisms in the field, and further investigation is needed to determine whether it can detect distinct differences in plankton density in the field. To address this, we analyzed eDNA in seawater samples collected at 1 km intervals at three depths over a linear distance of approximately 3.0 km in the Seto Inland Sea. The survey area included a location with a high density of Acoela (Praesagittifera naikaiensis). However, the eDNA signal for this was little to none, and its presence would not have been noticed if we did not have this information beforehand. Meanwhile, eDNA analysis enabled us to confirm the presence of a species of Placozoa that was previously undiscovered in the area. In summary, our results suggest that the number of sequence reads generated from eDNA samples in our project was not sufficient to predict the density of a particular species. However, eDNA can be useful for detecting organisms that have been overlooked using other methods.

Coherent directed movement toward food modeled in Trichoplax, a ciliated animal lacking a nervous system.

Trichoplax adhaerens is a small, ciliated marine animal that glides on surfaces grazing upon algae, which it digests externally. It has no muscles or nervous system and only six cell types, all but two of which are embedded in its epithelium. The epithelial cells are joined by apical adherens junctions; neither tight junctions nor gap junctions are present. Monociliated epithelial cells on the lower surface propel gliding. The cilia beat regularly, but asynchronously, and transiently contact the substrate with each stroke. The animal moves in random directions in the absence of food. We show here that it exhibits chemotaxis, moving preferentially toward algae embedded in a disk of agar. We present a mathematical model to explain how coherent, directional movements could arise from the collective actions of a set of ciliated epithelial cells, each independently sensing and responding to a chemoattractant gradient. The model incorporates realistic values for viscoelastic properties of cells and produces coordinated movements and changes in body shape that resemble the actual movements of the animal. The model demonstrates that an animal can move coherently in search of food without any need for chemical signaling between cells and introduces a different approach to modeling behavior in primitive multicellular organisms.

Hidden cell diversity in Placozoa: ultrastructural insights from Hoilungia hongkongensis.

From a morphological point of view, placozoans are among the most simple free-living animals. This enigmatic phylum is critical for our understanding of the evolution of animals and their cell types. Their millimeter-sized, disc-like bodies consist of only three cell layers that are shaped by roughly seven major cell types. Placozoans lack muscle cells and neurons but are able to move using their ciliated lower surface and take up food in a highly coordinated manner. Intriguingly, the genome of Trichoplax adhaerens, the founding member of the enigmatic phylum, has disclosed a surprising level of genetic complexity. Moreover, recent molecular and functional investigations have uncovered a much larger, so-far hidden cell-type diversity. Here, we have extended the microanatomical characterization of a recently described placozoan species-Hoilungia hongkongensis. In H. hongkongensis, we recognized the established canonical three-layered placozoan body plan but also came across several morphologically distinct and potentially novel cell types, among them novel gland cells and "shiny spheres"-bearing cells at the upper epithelium. Thus, the diversity of cell types in placozoans is indeed higher than anticipated.

Microchemical identification of enantiomers in early-branching animals: Lineage-specific diversification in the usage of D-glutamate and D-aspartate.

D-amino acids are unique and essential signaling molecules in neural, hormonal, and immune systems. However, the presence of D-amino acids and their recruitment in early animals is mostly unknown due to limited information about prebilaterian metazoans. Here, we performed the comparative survey of L-/D-aspartate and L-/D-glutamate in representatives of four phyla of early-branching Metazoa: cnidarians (Aglantha); placozoans (Trichoplax), sponges (Sycon) and ctenophores (Pleurobrachia, Mnemiopsis, Bolinopsis, and Beroe), which are descendants of ancestral animal lineages distinct from Bilateria. Specifically, we used high-performance capillary electrophoresis for microchemical assays and quantification of the enantiomers. L-glutamate and L-aspartate were abundant analytes in all species studied. However, we showed that the placozoans, cnidarians, and sponges had high micromolar concentrations of D-aspartate, whereas D-glutamate was not detectable in our assays. In contrast, we found that in ctenophores, D-glutamate was the dominant enantiomer with no or trace amounts of D-aspartate. This situation illuminates prominent lineage-specific diversifications in the recruitment of D-amino acids and suggests distinct signaling functions of these molecules early in the animal evolution. We also hypothesize that a deep ancestry of such recruitment events might provide some constraints underlying the evolution of neural and other signaling systems in Metazoa.

Sodium action potentials in placozoa: Insights into behavioral integration and evolution of nerveless animals.

Placozoa are small disc-shaped animals, representing the simplest known, possibly ancestral, organization of free-living animals. With only six morphological distinct cell types, without any recognized neurons or muscle, placozoans exhibit fast effector reactions and complex behaviors. However, little is known about electrogenic mechanisms in these animals. Here, we showed the presence of rapid action potentials in four species of placozoans (Trichoplax adhaerens [H1 haplotype], Trichoplax sp.[H2], Hoilungia hongkongensis [H13], and Hoilungia sp. [H4]). These action potentials are sodium-dependent and can be inducible. The molecular analysis suggests the presence of 5-7 different types of voltage-gated sodium channels, which showed substantial evolutionary radiation compared to many other metazoans. Such unexpected diversity of sodium channels in early-branched metazoan lineages reflect both duplication events and parallel evolution of unique behavioral integration in these nerveless animals.

Innate immunity in the simplest animals - placozoans.

BACKGROUND: Innate immunity provides the core recognition system in animals for preventing infection, but also plays an important role in managing the relationship between an animal host and its symbiont. Most of our knowledge about innate immunity stems from a few animal model systems, but substantial variation between metazoan phyla has been revealed by comparative genomic studies. The exploration of more taxa is still needed to better understand the evolution of immunity related mechanisms. Placozoans are morphologically the simplest organized metazoans and the association between these enigmatic animals and their rickettsial endosymbionts has recently been elucidated. Our analyses of the novel placozoan nuclear genome of Trichoplax sp. H2 and its associated rickettsial endosymbiont genome clearly pointed to a mutualistic and co-evolutionary relationship. This discovery raises the question of how the placozoan holobiont manages symbiosis and, conversely, how it defends against harmful microorganisms. In this study, we examined the annotated genome of Trichoplax sp. H2 for the presence of genes involved in innate immune recognition and downstream signaling. RESULTS: A rich repertoire of genes belonging to the Toll-like and NOD-like receptor pathways, to scavenger receptors and to secreted fibrinogen-related domain genes was identified in the genome of Trichoplax sp. H2. Nevertheless, the innate immunity related pathways in placozoans deviate in several instances from well investigated vertebrates and invertebrates. While true Toll- and NOD-like receptors are absent, the presence of many genes of the downstream signaling cascade suggests at least primordial Toll-like receptor signaling in Placozoa. An abundance of scavenger receptors, fibrinogen-related domain genes and Apaf-1 genes clearly constitutes an expansion of the immunity related gene repertoire specific to Placozoa. CONCLUSIONS: The found wealth of immunity related genes present in Placozoa is surprising and quite striking in light of the extremely simple placozoan body plan and their sparse cell type makeup. Research is warranted to reveal how Placozoa utilize this immune repertoire to manage and maintain their associated microbiota as well as to fend-off pathogens.

Insights into the evolution of digestive systems from studies of Trichoplax adhaerens.

Trichoplax, a member of the phylum Placozoa, is a tiny ciliated marine animal that glides on surfaces feeding on algae and cyanobacteria. It stands out from other animals in that it lacks an internal digestive system and, instead, digests food trapped under its lower surface. Here we review recent work on the phenotypes of its six cell types and their roles in digestion and feeding behavior. Phylogenomic analyses place Placozoa as sister to Eumetazoa, the clade that includes Cnidaria and Bilateria. Comparing the phenotypes of cells in Trichoplax to those of cells in the digestive epithelia of Eumetazoa allows us to make inferences about the cell types and mode of feeding of their ancestors. From our increasingly mechanistic understanding of feeding in Trichoplax, we get a glimpse into how primitive animals may have hunted and consumed food prior to the evolution of neurons, muscles, and internal digestive systems.

A non-bilaterian perspective on the development and evolution of animal digestive systems.

Digestive systems and extracellular digestion are key animal features, but their emergence during early animal evolution is currently poorly understood. As the last common ancestor of non-bilaterian animal groups (sponges, ctenophores, placozoans and cnidarians) dates back to the beginning of animal life, their study and comparison provides important insights into the early evolution of digestive systems and functions. Here, I have compiled an overview of the development and cell biology of digestive tissues in non-bilaterian animals. I will highlight the fundamental differences between extracellular and intracellular digestive processes, and how these are distributed among animals. Cnidarians (e.g. sea anemones, corals, jellyfish), the phylogenetic outgroup of bilaterians (e.g. vertebrates, flies, annelids), occupy a key position to reconstruct the evolution of bilaterian gut evolution. A major focus will therefore lie on the development and cell biology of digestive tissues in cnidarians, especially sea anemones, and how they compare to bilaterian gut tissues. In that context, I will also review how a recent study on the gastrula fate map of the sea anemone Nematostella vectensis challenges our long-standing conceptions on the evolution of cnidarian and bilaterian germ layers and guts.

Characterization of mitochondrial proteomes of nonbilaterian animals.

Mitochondria require ~1,500 proteins for their maintenance and proper functionality, which constitute the mitochondrial proteome (mt-proteome). Although a few of these proteins, mostly subunits of the electron transport chain complexes, are encoded in mitochondrial DNA (mtDNA), the vast majority are encoded in the nuclear genome and imported to the organelle. Previous studies have shown a continuous and complex evolution of mt-proteome among eukaryotes. However, there was less attention paid to mt-proteome evolution within Metazoa, presumably because animal mtDNA and, by extension, animal mitochondria are often considered to be uniform. In this analysis, two bioinformatic approaches (Orthologue-detection and Mitochondrial Targeting Sequence prediction) were used to identify mt-proteins in 23 species from four nonbilaterian phyla: Cnidaria, Ctenophora, Placozoa, and Porifera, as well as two choanoflagellates, the closest animal relatives. Our results revealed a large variation in mt-proteome in nonbilaterian animals in size and composition. Myxozoans, highly reduced cnidarian parasites, possessed the smallest inferred mitochondrial proteomes, while calcareous sponges possessed the largest. About 513 mitochondrial orthologous groups were present in all nonbilaterian phyla and human. Interestingly, 42 human mitochondrial proteins were not identified in any nonbilaterian species studied and represent putative innovations along the bilaterian branch. Several of these proteins were involved in apoptosis and innate immunity, two processes known to evolve within Metazoa. Conversely, several proteins identified as mitochondrial in nonbilaterian phyla and animal outgroups were absent in human, representing cases of possible loss. Finally, a few human cytosolic proteins, such as histones and cytosolic ribosomal proteins, were predicted to be targeted to mitochondria in nonbilaterian animals. Overall, our analysis provides the first step in characterization of mt-proteomes in nonbilaterian animals and understanding evolution of animal mt-proteome. © 2018 IUBMB Life, 70(12):1289-1301, 2018.

Neuropeptidergic integration of behavior in Trichoplax adhaerens, an animal without synapses.

Trichoplax adhaerens is a flat, millimeter-sized marine animal that adheres to surfaces and grazes on algae. Trichoplax displays a repertoire of different feeding behaviors despite the apparent absence of a true nervous system with electrical or chemical synapses. It glides along surfaces to find food, propelled by beating cilia on cells at its ventral surface, and pauses during feeding by arresting ciliary beating. We found that when endomorphin-like peptides are applied to an animal, ciliary beating is arrested, mimicking natural feeding pauses. Antibodies against these neuropeptides label cells that express the neurosecretory proteins and voltage-gated calcium channels implicated in regulated secretion. These cells are embedded in the ventral epithelium, where they comprise only 4% of the total, and are concentrated around the edge of the animal. Each bears a cilium likely to be chemosensory and used to detect algae. Trichoplax pausing during feeding or spontaneously in the absence of food often induce their neighbors to pause as well, even neighbors not in direct contact. Pausing behavior propagates from animal to animal across distances much greater than the signal that diffuses from just one animal, so we presume that the peptides secreted from one animal elicit secretion from nearby animals. Signal amplification by peptide-induced peptide secretion explains how a small number of sensory secretory cells lacking processes and synapses can evoke a wave of peptide secretion across the entire animal to globally arrest ciliary beating and allow pausing during feeding.

Retracing the path of planar cell polarity.

BACKGROUND: The Planar Cell Polarity pathway (PCP) has been described as the main feature involved in patterning cell orientation in bilaterian tissues. Recently, a similar phenomenon was revealed in cnidarians, in which the inhibition of this pathway results in the absence of cilia orientation in larvae, consequently proving the functional conservation of PCP signaling between Cnidaria and Bilateria. Nevertheless, despite the growing accumulation of databases concerning basal lineages of metazoans, very few information concerning the existence of PCP components have been gathered outside of Bilateria and Cnidaria. Thus, the origin of this module or its prevalence in early emerging metazoans has yet to be elucidated. RESULTS: The present study addresses this question by investigating the genomes and transcriptomes from all poriferan lineages in addition to Trichoplax (Placozoa) and Mnemiopsis (Ctenophora) genomes for the presence of the core components of this pathway. Our results confirm that several PCP components are metazoan innovations. In addition, we show that all members of the PCP pathway, including a bona fide Strabismus ortholog (Van gogh), are retrieved only in one sponge lineage (Homoscleromorpha) out of four. This highly suggests that the full PCP pathway dates back at least to the emergence of homoscleromorph sponges. Consequently, several secondary gene losses would have occurred in the three other poriferan lineages including Amphimedon queenslandica (Demospongiae). Several proteins were not retrieved either in placozoans or ctenophores leading us to discuss the difficulties to predict orthologous proteins in basally branching animals. Finally, we reveal how the study of multigene families may be helpful to unravel the relationships at the base of the metazoan tree. CONCLUSION: The PCP pathway antedates the radiation of Porifera and may have arisen in the last common ancestor of animals. Oscarella species now appear as key organisms to understand the ancestral function of PCP signaling and its potential links with Wnt pathways.

Bioinformatic prediction of Trichoplax adhaerens regulatory peptides.

Trichoplax adhaerens (phylum Placozoa) is a very simple organism that lacks a nervous system. However, its genome contains many genes essential for neuronal function and development. I report the results of regulatory peptide predictions for this enigmatic animal. Extensive transcriptome, genome, and predicted proteome mining allowed us to predict four insulins, at least five short peptide precursors, one granulin, one paracrine regulator of cell growth, and one complex temptin-attractin pheromone signaling system. The expression of three insulins, four short peptide precursors, granulin, and one out of the six temptin genes was detected. Five predicted regulatory peptide precursors could potentially release over 60 different mature peptides. Some of the predicted peptides are somewhat similar to anthozoan RW amides, Aplysia pedal peptide 3, and PRQFV amide. Other predicted short peptides could not readily be classified into established families. These data provide the foundation for the molecular, biochemical, physiological, and behavioral studies of one the most primitive animal coordination systems, and give unique insight into the origins and early evolution of the nervous system.

Dynamic evolution of mitochondrial ribosomal proteins in Holozoa.

We studied the highly dynamic evolution of mitochondrial ribosomal proteins (MRPs) in Holozoa. Most major clades within Holozoa are characterized by gains and/or losses of MRPs. The usefulness of gains of MRPs as rare genomic changes in phylogenetics is undermined by the high frequency of secondary losses. However, phylogenetic analyses of the MRP sequences provide evidence for the Acrosomata hypothesis, a sister group relationship between Ctenophora and Bilateria. An extensive restructuring of the mitochondrial genome and, as a consequence, of the mitochondrial ribosomes occurred in the ancestor of metazoans. The last MRP genes encoded in the mitochondrial genome were either moved to the nuclear genome or were lost. The strong decrease in size of the mitochondrial genome was probably caused by selection for rapid replication of mitochondrial DNA during oogenesis in the metazoan ancestor. A phylogenetic analysis of MRPL56 sequences provided evidence for a horizontal gene transfer of the corresponding MRP gene between metazoans and Dictyostelidae (Amoebozoa). The hypothesis that the requisition of additional MRPs compensated for a loss of rRNA segments in the mitochondrial ribosomes is corroborated by a significant negative correlation between the number of MRPs and length of the rRNA. Newly acquired MRPs evolved faster than bacterial MRPs and positions in eukaryote-specific MRPs were more strongly affected by coevolution than positions in prokaryotic MRPs in accordance with the necessity to fit these proteins into the pre-existing structure of the mitoribosome.

Parallel evolution of gravity sensing.

Omnipresent gravity affects all living organisms; it was a vital factor in the past and the current bottleneck for future space exploration. However, little is known about the evolution of gravity sensing and the comparative biology of gravity reception. Here, by tracing the parallel evolution of gravity sensing, we encounter situations when assemblies of homologous modules result in the emergence of non-homologous structures with similar systemic properties. This is a perfect example to study homoplasy at all levels of biological organization. Apart from numerous practical implementations for bioengineering and astrobiology, the diversity of gravity signaling presents unique reference paradigms to understand hierarchical homology transitions to the convergent evolution of integrative systems. Second, by comparing gravisensory systems in major superclades of basal metazoans (ctenophores, sponges, placozoans, cnidarians, and bilaterians), we illuminate parallel evolution and alternative solutions implemented by basal metazoans toward spatial orientation, focusing on gravitational sensitivity and locomotory integrative systems.

Brief History of Ctenophora.

Ctenophores are the descendants of the earliest surviving lineage of ancestral metazoans, predating the branch leading to sponges (Ctenophore-first phylogeny). Emerging genomic, ultrastructural, cellular, and systemic data indicate that virtually every aspect of ctenophore biology as well as ctenophore development are remarkably different from what is described in representatives of other 32 animal phyla. The outcome of this reconstruction is that most system-level components associated with the ctenophore organization result from convergent evolution. In other words, the ctenophore lineage independently evolved as high animal complexities with the astonishing diversity of cell types and structures as bilaterians and cnidarians. Specifically, neurons, synapses, muscles, mesoderm, through gut, sensory, and integrative systems evolved independently in Ctenophora. Rapid parallel evolution of complex traits is associated with a broad spectrum of unique ctenophore-specific molecular innovations, including alternative toolkits for making an animal. However, the systematic studies of ctenophores are in their infancy, and deciphering their remarkable morphological and functional diversity is one of the hot topics in biological research, with many anticipated surprises.