Early Branching Metazoan Research Articles

The genomes of the aquarium sponges Tethya wilhelma and Tethya minuta (Porifera: Demospongiae).

Sponges (Phylum Porifera) are aquatic sessile metazoans found worldwide in marine and freshwater environments. They are significant in the animal tree of life as one of the earliest-branching metazoan lineages and as filter feeders play crucial ecological roles, particularly in coral reefs, but are susceptible to the effects of climate change. In the face of the current biodiversity crisis, genomic data is crucial for species conservation efforts and predicting their evolutionary potential in response to environmental changes. However, there is a limited availability of culturable sponge species with annotated high-quality genomes to further comprehensive insights into animal evolution, function, and their response to the ongoing global change. Despite the publication of a few high-quality annotated sponge genomes, there remains a gap in resources for culturable sponge species. To address this gap, we provide high quality draft genomes of the two congeneric aquarium species Tethya wilhelma and Tethya minuta, small ball-shaped demosponges that are easily maintained long-term in ex situ culture. As such, they offer promising opportunities as laboratory models to contribute to advancing our understanding of sponge biology and provide valuable resources for studying animal evolution, function, and responses to environmental challenges.

The genomes of the aquarium sponges Tethya wilhelma and Tethya minuta (Porifera: Demospongiae)

Sponges (Phylum Porifera) are aquatic sessile metazoans found worldwide in marine and freshwater environments. They are significant in the animal tree of life as one of the earliest-branching metazoan lineages and as filter feeders play crucial ecological roles, particularly in coral reefs, but are susceptible to the effects of climate change. In the face of the current biodiversity crisis, genomic data is crucial for species conservation efforts and predicting their evolutionary potential in response to environmental changes. However, there is a limited availability of culturable sponge species with annotated high-quality genomes to further comprehensive insights into animal evolution, function, and their response to the ongoing global change. Despite the publication of a few high-quality annotated sponge genomes, there remains a gap in resources for culturable sponge species. To address this gap, we provide high quality draft genomes of the two congeneric aquarium species Tethya wilhelma and Tethya minuta, small ball-shaped demosponges that are easily maintained long-term in ex situ culture. As such, they offer promising opportunities as laboratory models to contribute to advancing our understanding of sponge biology and provide valuable resources for studying animal evolution, function, and responses to environmental challenges.

Illustrated Neuroanatomy of Ctenophores: Immunohistochemistry.

Ctenophores or comb jellies are representatives of an enigmatic lineage of early branching metazoans with complex tissue and organ organization. Their biology and even microanatomy are not well known for most of these fragile pelagic and deep-water species. Here, we present immunohistochemical protocols successfully tested on more than a dozen ctenophores. This chapter also illustrates neural organization in several reference species of the phylum (Pleurobrachia bachei, P. pileus, Mnemiopsis leidyi, Bolinopsis microptera, Beroe ovata, and B. abyssicola) as well as numerous ciliated structures in different functional systems. The applications of these protocols illuminate a very complex diversification of cell types comparable to many bilaterian lineages.

Gene Expression Patterns in the Ctenophore Pleurobrachia bachei: In Situ Hybridization.

In situ hybridization is a powerful and precise tool for revealing cell- and tissue-specific gene expression and a critical approach to validating single-cell RNA-seq (scRNA-seq). However, applying it to highly fragile animals such as ctenophores is challenging. Here, we present an in situ hybridization protocol for adult Pleurobrachia bachei (Cydippida)-a notable reference species representing the earliest-branching metazoan lineage, Ctenophora, sister to the rest of Metazoa. We provided expression patterns for several markers of cell phenotypes, as illustrated examples. The list includes predicted small secretory molecules/neuropeptides, WntX, genes encoding RNA-binding proteins (Musashi, Elav, Dicer, Argonaut), Neuroglobin, and selected transcription factors such as BarX. Both cell- and organ-specific expression of these genes further support the convergent evolution of many ctenophore innovations, which are remarkably distinct from tissue and organ specification in other basal metazoan lineages.

Long-Term Culturing of Placozoans (Trichoplax and Hoilungia).

The phylum Placozoa remains one of the least explored among early-branching metazoan lineages. For over 130years, this phylum had been represented by the single species Trichoplax adhaerens-an animal with the simplest known body plan (three cell layers without any organs) but complex behaviors. Recently, extensive sampling of placozoans across the globe and their subsequent genetic analysis have revealed incredible biodiversity with numerous cryptic species worldwide. However, only a few culture protocols are available to date, and all are for one species only. Here, we describe the breeding of four different species representing two placozoan genera: Trichoplax adhaerens, Trichoplax sp. H2, Hoilungia sp. H4, and Hoilungia hongkongensis originating from diverse biotopes. Our protocols allow to culture all species under comparable conditions. Next, we outlined various food sources and optimized strain-specific parameters enabling long-term culturing. These protocols can facilitate comparative analyses of placozoan biology and behaviors, which together will contribute to deciphering general principles of animal organization.

Distinct stem-like cell populations facilitate functional regeneration of the Cladonema medusa tentacle.

Blastema formation is a crucial process that provides a cellular source for regenerating tissues and organs. While bilaterians have diversified blastema formation methods, its mechanisms in non-bilaterians remain poorly understood. Cnidarian jellyfish, or medusae, represent early-branching metazoans that exhibit complex morphology and possess defined appendage structures highlighted by tentacles with stinging cells (nematocytes). Here, we investigate the mechanisms of tentacle regeneration, using the hydrozoan jellyfish Cladonema pacificum. We show that proliferative cells accumulate at the tentacle amputation site and form a blastema composed of cells with stem cell morphology. Nucleoside pulse-chase experiments indicate that most repair-specific proliferative cells (RSPCs) in the blastema are distinct from resident stem cells. We further demonstrate that resident stem cells control nematogenesis and tentacle elongation during both homeostasis and regeneration as homeostatic stem cells, while RSPCs preferentially differentiate into epithelial cells in the newly formed tentacle, analogous to lineage-restricted stem/progenitor cells observed in salamander limbs. Taken together, our findings propose a regeneration mechanism that utilizes both resident homeostatic stem cells (RHSCs) and RSPCs, which in conjunction efficiently enable functional appendage regeneration, and provide novel insight into the diversification of blastema formation across animal evolution.

Regeneration in calcareous sponge relies on 'purse-string' mechanism and the rearrangements of actin cytoskeleton.

The crucial step in any regeneration process is epithelization, i.e. the restoration of anepithelium structural and functional integrity. Epithelization requires cytoskeletal rearrangements, primarily of actin filaments and microtubules. Sponges (phylum Porifera) are early branching metazoans with pronounced regenerative abilities. Calcareous sponges have a unique step during regeneration: theformation of a temporary structure, calledregenerative membrane which initially covers a wound. It forms due to the morphallactic rearrangements of exopinaco- and choanoderm epithelial-like layers. The current study quantitatively evaluates morphological changes and characterises underlying actin cytoskeleton rearrangements during regenerative membrane formation in asconoid calcareous sponge Leucosolenia variabilis through a combination of time-lapse imaging, immunocytochemistry, and confocal laser scanning microscopy. Regenerative membrane formation has non-linear stochastic dynamics with numerous fluctuations. The pinacocytes at the leading edge of regenerative membrane form a contractile actomyosin cable. Regenerative membrane formation either depends on its contraction or being coordinated through it. The cell morphology changes significantly during regenerative membrane formation. Exopinacocytes flatten, their area increases, while circularity decreases. Choanocytes transdifferentiate into endopinacocytes, losing microvillar collar and flagellum. Their area increases and circularity decreases. Subsequent redifferentiation of endopinacocytes into choanocytes is accompanied by inverse changes in cell morphology. All transformations rely on actin filament rearrangements similar to those characteristic of bilaterian animals. Altogether, we provide here a qualitative and quantitative description of cell transformations during reparative epithelial morphogenesis in a calcareous sponge.

Recording cilia activity in ctenophores: effects of nitric oxide and low molecular weight transmitters.

Cilia are the major effectors in Ctenophores, but very little is known about their transmitter control and integration. Here, we present a simple protocol to monitor and quantify cilia activity and provide evidence for polysynaptic control of cilia coordination in ctenophores. We also screened the effects of several classical bilaterian neurotransmitters (acetylcholine, dopamine, L-DOPA, serotonin, octopamine, histamine, gamma-aminobutyric acid (GABA), L-aspartate, L-glutamate, glycine), neuropeptide (FMRFamide), and nitric oxide (NO) on cilia beating in Pleurobrachia bachei and Bolinopsis infundibulum. NO and FMRFamide produced noticeable inhibitory effects on cilia activity, whereas other tested transmitters were ineffective. These findings further suggest that ctenophore-specific neuropeptides could be major candidates for signal molecules controlling cilia activity in representatives of this early-branching metazoan lineage.

Nitric oxide signaling in ctenophores.

Nitric oxide (NO) is one of the most ancient and versatile signal molecules across all domains of life. NO signaling might also play an essential role in the origin of animal organization. Yet, practically nothing is known about the distribution and functions of NO-dependent signaling pathways in representatives of early branching metazoans such as Ctenophora. Here, we explore the presence and organization of NO signaling components using Mnemiopsis and kin as essential reference species. We show that NO synthase (NOS) is present in at least eight ctenophore species, including Euplokamis and Coeloplana, representing the most basal ctenophore lineages. However, NOS could be secondarily lost in many other ctenophores, including Pleurobrachia and Beroe. In Mnemiopsis leidyi, NOS is present both in adult tissues and differentially expressed in later embryonic stages suggesting the involvement of NO in developmental mechanisms. Ctenophores also possess soluble guanylyl cyclases as potential NO receptors with weak but differential expression across tissues. Combined, these data indicate that the canonical NO-cGMP signaling pathways existed in the common ancestor of animals and could be involved in the control of morphogenesis, cilia activities, feeding and different behaviors.

Alternative neural systems: What is a neuron? (Ctenophores, sponges and placozoans).

How to make a neuron, a synapse, and a neural circuit? Is there only one 'design' for a neural architecture with a universally shared genomic blueprint across species? The brief answer is "No." Four early divergent lineages from the nerveless common ancestor of all animals independently evolved distinct neuroid-type integrative systems. One of these is a subset of neural nets in comb jellies with unique synapses; the second lineage is the well-known Cnidaria + Bilateria; the two others are non-synaptic neuroid systems in sponges and placozoans. By integrating scRNA-seq and microscopy data, we revise the definition of neurons as synaptically-coupled polarized and highly heterogenous secretory cells at the top of behavioral hierarchies with learning capabilities. This physiological (not phylogenetic) definition separates 'true' neurons from non-synaptically and gap junction-coupled integrative systems executing more stereotyped behaviors. Growing evidence supports the hypothesis of multiple origins of neurons and synapses. Thus, many non-bilaterian and bilaterian neuronal classes, circuits or systems are considered functional rather than genetic categories, composed of non-homologous cell types. In summary, little-explored examples of convergent neuronal evolution in representatives of early branching metazoans provide conceptually novel microanatomical and physiological architectures of behavioral controls in animals with prospects of neuro-engineering and synthetic biology.

A developmental role for the chromatin-regulating CoREST complex in the cnidarian Nematostella vectensis

BackgroundChromatin-modifying proteins are key players in the regulation of development and cell differentiation in animals. Most chromatin modifiers, however, predate the evolution of animal multicellularity, and how they gained new functions and became integrated into the regulatory networks underlying development is unclear. One way this may occur is the evolution of new scaffolding proteins that integrate multiple chromatin regulators into larger complexes that facilitate coordinated deposition or removal of different chromatin modifications. We test this hypothesis by analyzing the evolution of the CoREST-Lsd1-HDAC complex.ResultsUsing phylogenetic analyses, we show that a bona fide CoREST homolog is found only in choanoflagellates and animals. We then use the sea anemone Nematostella vectensis as a model for early branching metazoans and identify a conserved CoREST complex by immunoprecipitation and mass spectrometry of an endogenously tagged Lsd1 allele. In addition to CoREST, Lsd1 and HDAC1/2 this complex contains homologs of HMG20A/B and PHF21A, two subunits that have previously only been identified in mammalian CoREST complexes. NvCoREST expression overlaps fully with that of NvLsd1 throughout development, with higher levels in differentiated neural cells. NvCoREST mutants, generated using CRISPR-Cas9, fail to develop beyond the primary polyp stage, thereby revealing essential roles during development and for the differentiation of cnidocytes that phenocopy NvLsd1 mutants. We also show that this requirement is cell autonomous using a cell-type-specific rescue approach.ConclusionsThe identification of a Nematostella CoREST-Lsd1-HDAC1/2 complex, its similarity in composition with the vertebrate complex, and the near-identical expression patterns and mutant phenotypes of NvCoREST and NvLsd1 suggest that the complex was present before the last common cnidarian-bilaterian ancestor and thus represents an ancient component of the animal developmental toolkit.

Tissue homeostasis in sponges: Quantitative analysis of cell proliferation and apoptosis.

Tissues of multicellular animals are maintained due to a tight balance between cell proliferation and programmed cell death. Sponges are early branching metazoans essential to understanding the key mechanisms of tissue homeostasis. This article is dedicated to the comparative analysis of proliferation and apoptosis in intact tissues of two sponges, Halisarca dujardinii (class Demospongiae) and Leucosolenia variabilis (class Calcarea). Labeled nucleotides EdU and anti-phosphorylated histone 3 antibodies reveal a considerable number of cycling cells in intact tissues of both species. Quantitative DNA staining reveals the classic cell cycle distribution curve. The main type of cycling cells are choanocytes - flagellated cells of the aquiferous system. The rate of proliferation remains constant throughout various areas of sponge bodies that contain choanocytes. The EdU tracking experiments conducted in H. dujardinii indicate that choanocytes may give rise to mesohyl cells through migration. The number of apoptotic cells in tissues of both species is insignificant, although being comparable to the renewing tissues of other animals. In vivo studies with tetramethylrhodamine ethyl ester and CellEvent Caspase-3/7 indicate that apoptosis might be independent of mitochondrial outer membrane permeabilization. Altogether, a combination of confocal laser scanning microscopy and flow cytometry provides a quantitative description of cell proliferation and apoptosis in sponges displaying either rapid growth or cell turnover.

Oogenesis and lipid metabolism in the deep-sea sponge Phakellia ventilabrum (Linnaeus, 1767)

Sponges contain an astounding diversity of lipids that serve in several biological functions, including yolk formation in their oocytes and embryos. The study of lipid metabolism during reproduction can provide information on food-web dynamics and energetic needs of the populations in their habitats, however, there are no studies focusing on the lipid metabolism of sponges during their seasonal reproduction. In this study, we used histology, lipidome profiling (UHPLC-MS), and transcriptomic analysis (RNA-seq) on the deep-sea sponge Phakellia ventilabrum (Demospongiae, Bubarida), a key species of North-Atlantic sponge grounds, with the goal to (i) assess the reproductive strategy and seasonality of this species, (ii) examine the relative changes in the lipidome signal and the gene expression patterns of the enzymes participating in lipid metabolism during oogenesis. Phakellia ventilabrum is an oviparous and most certainly gonochoristic species, reproducing in May and September in the different studied areas. Half of the specimens were reproducing, generating two to five oocytes per mm2. Oocytes accumulated lipid droplets and as oogenesis progressed, the signal of most of the unsaturated and monounsaturated triacylglycerides increased, as well as of a few other phospholipids. In parallel, we detected upregulation of genes in female tissues related to triacylglyceride biosynthesis and others related to fatty acid beta-oxidation. Triacylglycerides are likely the main type of lipid forming the yolk in P. ventilabrum since this lipid category has the most marked changes. In parallel, other lipid categories were engaged in fatty acid beta-oxidation to cover the energy requirements of female individuals during oogenesis. In this study, the reproductive activity of the sponge P. ventilabrum was studied for the first time uncovering their seasonality and revealing 759 lipids, including 155 triacylglycerides. Our study has ecological and evolutionary implications providing essential information for understanding the molecular basis of reproduction and the origins and formation of lipid yolk in early-branching metazoans.

Deep-water fossil assemblages from the Ediacaran-Cambrian transition of western Hunan, South China and their biostratigraphic and evolutionary implications

The Ediacaran-Cambrian (E-C) transition was a critical stage for the origin and evolution of early complex life on Earth. Because studies of E-C biotas and environments are relatively fewer for deep-water than for shallow-water facies, we undertook a systematic paleontological analysis of the E-C transition in the deep-water Liuchapo Formation at Longbizui, South China. Two sequential but distinct assemblages were recovered, namely the Lower Liuchapo Assemblage (LLA) and the Upper Liuchapo Assemblage (ULA). The LLA consists mainly of soft-bodied fossils, including Longbizuiella hunanensis gen. et sp. nov., Nenoxites jishouensis sp. nov., Nenoxites irregularis sp. nov., Nenoxites sp., Helanoichnus sp., Shaanxilithes ? sp. and tiny-sized spherical fossils. The ULA is dominated by skeletal organisms comprising the small shelly faunas (SSFs, mainly protoconodonts, hyoliths and Cambroscleritida), sponge spicules, and the first reported Poratusiramus xiangxiensis gen. et sp. nov. Biostratigraphic correlation support a latest Ediacaran age for the LLA, whereas the majority of the ULA belongs to the early Cambrian—thus the change from the LLA to the ULA coincided with the E-C transition. Our results suggest that, during the E-C transition, a biological revolution may have occurred in deep-water settings synchronously with the well-studied faunal turnover in shallow-water environments. At Longbizui, fossil abundance and diversity decreased gradually during the latest Ediacaran and recovered stepwise after the E-C transition. The deep-water fauna, including both benthic and pelagic members, developed a biomineralization ability during the E-C transition. As calcareous and possibly phosphatic skeletal organisms began to flourish in shallow-water environments, siliceous skeleton-forming organisms, represented by the spicule-bearing sponges, colonized the deep seafloor in great abundance. In addition, the siliceous spicules documented in this study record fast evolutionary development during the early history of sponges, one of the earliest-branching metazoan phyla. The evolution of spicules from monaxon to diaxon-triaxon and then to tetraxon and polyactinal forms, as well as their considerable increase in diameter and length, was achieved during the first SSF biozone. • First detailed study of deep-water fossil assemblages of Ediacaran-Cambrian age. • Deep-water fauna may have experienced a turnover during the E -C transition. • Patterns of biodiversity and abundance paralleled those of shallow-marine habitats. • Record of diversification of earliest sponges and other siliceous biomineralizers. • Utility of evolutionary sequence of sponge spicule types as biostratigraphic index.

Molecular mechanisms of sperm motility are conserved in an early-branching metazoan

Efficient and targeted sperm motility is essential for animal reproductive success. Sperm from mammals and echinoderms utilize a highly conserved signaling mechanism in which sperm motility is stimulated by pH-dependent activation of the cAMP-producing enzyme soluble adenylyl cyclase (sAC). However, the presence of this pathway in early-branching metazoans has remained unexplored. Here, we found that elevating cytoplasmic pH induced a rapid burst of cAMP signaling and triggered the onset of motility in sperm from the reef-building coral Montipora capitata in a sAC-dependent manner. Expression of sAC in the mitochondrial-rich midpiece and flagellum of coral sperm support a dual role for this molecular pH sensor in regulating mitochondrial respiration and flagellar beating and thus motility. In addition, we found that additional members of the homologous signaling pathway described in echinoderms, both upstream and downstream of sAC, are expressed in coral sperm. These include the Na+/H+ exchanger SLC9C1, protein kinase A, and the CatSper Ca2+ channel conserved even in mammalian sperm. Indeed, the onset of motility corresponded with increased protein kinase A activity. Our discovery of this pathway in an early-branching metazoan species highlights the ancient origin of the pH-sAC-cAMP signaling node in sperm physiology and suggests that it may be present in many other marine invertebrate taxa for which sperm motility mechanisms remain unexplored. These results emphasize the need to better understand the role of pH-dependent signaling in the reproductive success of marine animals, particularly as climate change stressors continue to alter the physiology of corals and other marine invertebrates.

Evidence for BMP-mediated specification of primordial germ cells in an indirect-developing hemichordate.

Primordial germ cells (PGCs) are specified during development by either one of two major mechanisms, the preformation mode or the inductive mode. Because the inductive mode is widely employed by many bilaterians and early branching metazoan lineages, it has been postulated as an ancestral mechanism. However, among the deuterostome species that have been studied, invertebrate chordates use the preformation mode, while many vertebrate and echinoderm species are known to utilize an inductive mechanism, thus leaving the evolutionary history of PGC specification in the deuterostome lineage unclear. Hemichordates are the sister phylum of echinoderms, and together they form a clade called Ambulacraria that represents the closest group to the chordates. Thus, research in hemichordates is highly informative for resolving this issue. In this study, we investigate the developmental process of PGCs in an indirect-developing hemichordate, Ptychodera flava. We show that maternal transcripts of the conserved germline markers vasa, nanos, and piwi1 are ubiquitously distributed in early P. flava embryos, and these genes are coexpressed specifically in the dorsal hindgut starting from the gastrula stage. Immunostaining revealed that Vasa protein is concentrated toward the vegetal pole in early P. flava embryos, and it is restricted to cells in the dorsal hindgut of gastrulae and newly hatched larvae. The Vasa-positive cells later contribute to the developing trunk coeloms of the larvae and eventually reside in the adult gonads. We further show that bone morphogenetic protein (BMP) signaling is required to activate expression of the germline determinants in the gastrula hindgut, suggesting that PGC specification is induced by BMP signaling in P. flava. Our data support the hypothesis that the inductive mode is a conserved mechanism in Ambulacraria, which might even trace back to the common ancestor of Deuterostomes.

Mitogenomics suggests a sister relationship of Relicanthus daphneae (Cnidaria: Anthozoa: Hexacorallia: incerti ordinis) with Actiniaria

Relicanthus daphneae (formerly Boloceroides daphneae) was first described in 2006 as a giant sea anemone based on morphology. In 2014, its classification was challenged based on molecular data: using five genes, Relicanthus was resolved sister to zoanthideans, but with mixed support. To better understand the evolutionary relationship of Relicanthus with other early-branching metazoans, we present 15 newly-sequenced sea anemone mitochondrial genomes and a mitogenome-based phylogeny including all major cnidarian groups, sponges, and placozoans. Our phylogenetic reconstruction reveals a moderately supported sister relationship between Relicanthus and the Actiniaria. Morphologically, the cnidae of Relicanthus has apical flaps, the only existing synapomorphy for sea anemones. Based on both molecular and morphological results, we propose a third suborder (Helenmonae) within the Actiniaria to accommodate Relicanthus. Although Relicanthus shares the same gene order and content with other available actiniarian mitogenomes, it is clearly distinct at the nucleotide level from anemones within the existing suborders. The phylogenetic position of Relicanthus could reflect its association with the periphery of isolated hydrothermal vents, which, although patchy and ephemeral, harbor unique chemosynthetic communities that provide a relatively stable food source to higher trophic levels over long evolutionary timescales. The ability to colonize the deep sea and the periphery of new vent systems may be facilitated by Relicanthus’ large and extremely yolky eggs.

Extracellular matrix and morphogenesis in cnidarians: a tightly knit relationship.

Cnidarians, members of an early-branching metazoan phylum, possess an extracellular matrix (ECM) between their two epithelial cell layers, called the mesoglea. The cnidarian ECM, which is best studied in Hydra, contains matrix components reflective of both interstitial matrix and basement membrane. The identification of core matrisome components in cnidarian genomes has led to the notion that the basic composition of vertebrate ECM is of highly conserved nature and can be traced back to pre-bilaterians. While in vertebrate classes ECM factors have often diverged and acquired specialized functions in the context of organ development, cnidarians with their simple body plan retained direct links between ECM and morphogenesis. Recent advances in genetic manipulation techniques have provided tools for systematically studying cnidarian ECM function in body axis patterning and regeneration.

The Challenge of the Sponge Suberites domuncula (Olivi, 1792) in the Presence of a Symbiotic Bacterium and a Pathogen Bacterium.

Sponges, which are in close contact with numerous bacteria in prey/predator, symbiotic and pathogenic relationships, must provide an appropriate response in such situations. This starts with a discriminating recognition of the partner either by a physical contact or through secreted molecules or both. We investigated the expression of the Toll-like receptor, Caspase 3/7, Tumor Necrosis Factor receptor-associated factor 6, Bcl-2 homology protein-2 and macrophage expressed genes of axenic sponge cells in the presence of a symbiotic bacterium (Endozoicomonas sp. Hex311), a pathogen bacterium (Pseudoalteromonas sp. 1A1), their exoproducts and lipopolysaccharides. The vast majority of answers are in line with what could be observed with the symbiotic bacterium. The pathogenic bacterium seems to profit from the eukaryotic cell: suppression of the production of the antibacterial compound, inhibition of the apoptosis caspase-dependent pathway, deregulation of bacterial recognition. This work contributes new scientific knowledge in the field of immunology and apoptosis in early branching metazoan harboring within its tissue and cells a large number of symbiotic bacteria.

Neuromuscular organization of the Ctenophore Pleurobrachia bachei.

Ctenophores are descendants of one of the earliest branching metazoan lineage with enigmatic nervous systems. The lack of convenient neurogenic molecules and neurotransmitters suggests an extensive parallel evolution and independent origins of neurons and synapses. However, the field lags due to the lack of microanatomical data about the neuro-muscular systems in this group of animals. Here, using immunohistochemistry and scanning electron microscopy, we describe the organization of both muscular and nervous systems in the sea gooseberry, Pleurobrachia bachei, from North Pacific. The diffuse neural system of Pleurobrachia consists of two subsystems: the subepithelial neural network and the mesogleal net with about 5,000-7,000 neurons combined. Our data revealed the unexpected complexity of neuromuscular organization in this basal metazoan lineage. The anatomical diversity of cell types includes at least nine broad categories of neurons, five families of surface receptors and more than two dozen types of muscle cells as well as regional concentrations of neuronal elements to support ctenophore feeding, complex swimming, escape, and prey capture behaviors. In summary, we recognize more than 80 total morphological cell types. Thus, in terms of cell-type specification and diversity, ctenophores significantly exceed what we currently know about other prebilaterian groups (placozoan, sponges, and cnidarians), and some basal bilaterians.