How animal cell types, tissues, and regional body plans arose is a fundamental question in EvoDevo. Many current efforts attempt to link genetic information to the morphology of cells, tissues and regionalization of animal body plans using single-cell sequencing of cell populations. However, a lack of in-depth understanding of the morphology of non-bilaterian animals remains a considerable block to understanding the transitions between bilaterian and non-bilaterian cells and tissues. Sponges (Porifera), one of the earliest diverging animal phyla, pose a particular challenge to this endeavour, because their body plans lack mouths, gut, conventional muscle and nervous systems. With a goal to help bridge this gap, we have studied the morphology, behaviour and transcriptomics of cells and tissue types of an easily accessible and well-studied species of freshwater sponge, Ephydatia muelleri. New features described here include: a polarized external epithelium, a new contractile sieve cell that forms the entry to incurrent canals, motile cilia on apopyle cells at the exit of choanocyte chambers, and non-motile cilia on cells in excurrent canals and oscula. Imaging cells in vivo shows distinct behavioural characteristics of motile cells in the mesohyl. Transcriptomic phenotypes of three cell types (cystencytes, choanocytes and archaeocytes) captured live indicate that cell-type transcriptomes are distinct. Importantly, individual archaeocytes show a range of transcriptomic phenotypes which is supported by the distinct expression of different genes by subsets of this cell type. In contrast, all five choanocyte cells sampled live revealed highly uniform transcriptomes with significantly fewer genes expressed than in other cell types. Our study shows that sponges have tissues whose morphology and cell diversity are both functionally complex, but which together enable the sponge, like other metazoans, to sense and respond to stimuli.

Freshwater sponges fulfill critical ecological functions, including maintaining water quality, regulating nutrient dynamics, offering habitats for diverse taxa, and serving as a vital food source for various species. However, their patterns of dispersal and genetic connectivity remain inadequately understood, posing significant challenges to effective conservation assessments. We examined genetic connectivity and genetic adaptation to local environmental conditions in Ephydatia muelleri across its geographic range using ddRADseq-derived SNPs from 106 individuals collected from 11 localities spanning North America, Europe, and Asia. Analysis of 3,182 neutral SNPs revealed low connectivity and strong genetic structure among regions within two main genetic clusters of North America and Eurasia, while 115 SNPs identified to be under selection showed considerable evidence for differentiated, polygenic adaptation to light and temperature conditions across sampled locations, as well as selection on gene regulatory processes. These findings align with the monopolization hypothesis, suggesting that historical climatic and geological conditions of the Last Glacial Maximum, including habitat expansion, contraction, and natural barriers, have contributed more to the current genetic structure of E. muelleri populations than contemporary gene flow, which is restricted by monopolistic habitat colonization by this species. Our results provide novel support for ecological theory on dispersal in aquatic invertebrates, as well as insights into the plasticity of E. muelleri in the face of varying environmental conditions that are fundamentally important for freshwater ecosystem conservation. ### Competing Interest Statement The authors have declared no competing interest.

In bilaterian animals, gene regulation is shaped by a combination of linear and spatial regulatory information. Regulatory elements along the genome are integrated into gene regulatory landscapes through chromatin compartmentalization1,2, insulation of neighbouring genomic regions3,4 and chromatin looping that brings together distal cis-regulatory sequences5. However, the evolution of these regulatory features is unknown because the three-dimensional genome architecture of most animal lineages remains unexplored6,7. To trace the evolutionary origins of animal genome regulation, here we characterized the physical organization of the genome in non-bilaterian animals (sponges, ctenophores, placozoans and cnidarians)8,9 and their closest unicellular relatives (ichthyosporeans, filastereans and choanoflagellates)10 by combining high-resolution chromosome conformation capture11,12 with epigenomic marks and gene expression data. Our comparative analysis showed that chromatin looping is a conserved feature of genome architecture in ctenophores, placozoans and cnidarians. These sequence-determined distal contacts involve both promoter-enhancer and promoter-promoter interactions. By contrast, chromatin loops are absent in the unicellular relatives of animals. Our findings indicate that spatial genome regulation emerged early in animal evolution. This evolutionary innovation introduced regulatory complexity, ultimately facilitating the diversification of animal developmental programmes and cell type repertoires.

Cilia are found on the epithelia of almost all metazoans, so their absence from the epithelia of all but one class of Porifera is puzzling. Homoscleromorph sponges possess ciliated epithelia, but their function and evolutionary history within Porifera are unclear. We compared the ciliary beat frequencies (CBFs) of cilia on outer epithelia of the homoscleromorph sponge Oscarella sp. with those of other animals to suggest possible functions for the cilia. Settled Stage 4 buds, or juveniles, were found to have a higher CBF than free-moving Stage 1 buds, and CBF was within the range of cilia that function in mucus transport in other aquatic invertebrates. Scanning Electron Microscopy (SEM) images of buds fixed with ruthenium red to detect the presence of mucus showed that mucus was associated with the cilia of the exopinacoderm and both SEM and immunofluorescence images revealed fields of homogeneously oriented cilia. Confocal imaging of fluorescent beads also showed that cilia beat in the same direction. Movement of beads was reduced by nocodazole treatment indicating that the movement of particles over the surface was caused by ciliary beat. These results suggest that cilia on the epithelia of Homoscleromorph sponges are involved in mucociliary-driven particle flux, and may be used to clean the surface using mucus.

ABSTRACT Sponges (phylum Porifera) are an early diverging animal lineage without nervous and muscular systems, and yet they are able to produce coordinated whole-body contractions in response to disturbances. Little is known about the underlying signalling mechanisms in coordinating such responses. Previous studies demonstrated that sponges respond specifically to chemicals such as l-glutamate and γ-amino-butyric acid (GABA), which trigger and prevent contractions, respectively. Genes for purinergic P2X-like receptors are present in several sponge genomes, leading us to ask whether ATP works with glutamate to coordinate contractions in sponges as it does in other animal nervous systems. Using pharmacological approaches on the freshwater sponge Ephydatia muelleri, we show that ATP is involved in coordinating contractions. Bath application of ATP caused a rapid, sustained expansion of the excurrent canals in a dose-dependent manner. Complete contractions occurred when ATP was added in the presence of apyrase, an enzyme that hydrolyses ATP. Application of ADP, the first metabolic product of ATP hydrolysis, triggered complete contractions, whereas AMP, the subsequent metabolite, did not trigger a response. Blocking ATP from binding and activating P2X receptors with pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) prevented both glutamate- and ATP-triggered contractions, suggesting that ATP works downstream of glutamate. Bioinformatic analysis revealed two P2X receptor sequences, one of which groups with other vertebrate P2X receptors. Altogether, our results confirm that purinergic signalling by ATP is involved in coordinating contractions in the freshwater sponge.

The freshwater sponge, Ephydatia muelleri, lacks a nervous or endocrine system and yet it exhibits a coordinated whole-body action known as a “sneeze” that can be triggered by exposure to l-glutamate. It is not known how l-glutamate is obtained by E. muelleri in sufficient quantities (i.e., 70 µM) to mediate this response endogenously. The present study tested the hypothesis that l-glutamate can be directly acquired from the environment across the body surface of E. muelleri. We demonstrate carrier mediated uptake of two distinct saturable systems with maximal transport rates (Jmax) of 64.27 ± 4.98 and 25.12 ± 1.87 pmols mg−1 min−1, respectively. The latter system has a higher calculated substrate affinity (Km) of 2.87 ± 0.38 µM compared to the former (8.75 ± 1.00 µM), indicative of distinct systems that can acquire l-glutamate at variable environmental concentrations. Further characterization revealed potential shared pathways of l-glutamate uptake with other negatively charged amino acids, namely d-glutamate and l-aspartate, as well as the neutral amino acid L-alanine. We demonstrate that l-glutamate uptake does not appear to rely on exogenous sodium or proton concentrations as removal of these ions from the bathing media did not significantly alter uptake. Likewise, l-glutamate uptake does not seem to rely on internal proton motive forces driven by VHA as application of 100 nM of the VHA inhibitor bafilomycin did not alter uptake rates within E. muelleri tissues. Whether the acquired amino acid is used to supplement feeding or is stored and accumulated to mediate the sneeze response remains to be determined.

Sponges are remarkably active animals with a range of behaviors that are broadly termed sneezes. Little is known about what triggers sneezes or how sneezes propagate in an animal that lacks either muscular or nervous systems. We explored the triggers of sponge behavior in situ in the demosponge Suberites concinnus. The placement of this tennis ball-sized sponge at the focal point of a camera array on an instrument platform in Barkley Sound, BC, Canada, provided a unique opportunity for our investigation. The observatory has a suite of environmental sensors measuring parameters such as photosynthetically active radiation (PAR), oxygen, turbidity, salinity, pressure, current speed, and temperature. An array of 8 cameras designed for 3D imaging was used to capture hourly images. Our findings revealed both long-term (annual) and short-term (daily) behaviors in S. concinnus. The sponge contracted for 2 mo each winter. In February, it gradually expanded and became very active with short-term full-body contractions each lasting 11 h through the summer. A meta-analysis of contraction behaviors in other species of sponge showed that contraction duration is a factor of sponge size and that all sponges contract faster than they expand. Invertebrates and fish were frequently in contact with the sponge, but no single interaction was found to correlate with patterns of sponge behavior. However, significant correlations between sponge behaviors and chlorophyll and turbidity were found using generalized additive mixed models. Our study underscores the dynamic nature of these sessile filter feeders and their responses to their environment.

Reproduction is a key step for propagation of any species. Consequently, gametogenesis is crucial, as it links one generation to the other. Oogenesis is influenced by different factors, but it is usually related to the quality and quantity of the food and the capacity of the female to convert these resources into egg production. In Demospongiae (Porifera), oocytes vary in several aspects (e.g., origin, size, and vitellogenic pathways). However, data on oocyte morphology is still fragmentary, and the ultrastructural organization of reproductive cells has been investigated only in a few species, mainly of viviparous sponges. Here, we aimed to comprehend the oogenesis of two tropical oviparous demosponges (Cinachyrella apion and Tethya maza) using light and electron microscopy. In both species, oocytes seemed to originate from archaeocytes. Oocytes of C. apion were surrounded by a collagenous matrix and nurse cells containing many lipid vesicles. The increase of biosynthetic organelles, concomitantly with the presence of yolk vesicle in the ooplasm, indicated that the vitellogenesis was carried out through the mixed pathway. The oocytes of T. maza were surrounded by a follicle cell membrane and nurse cells containing yolk vesicles. The absence of characteristic biosynthetic organelles in the egg of this species indicated that vitellogenesis occured through the heterosynthetic pathway. The oogenesis of C. apion is similar to other species of the genus, while the follicle membrane and nurse cells surrounding the oocytes of T. maza are not observed in any other species of Tethya. These accessory cells were considered to have a trophic role during the oogenesis of the studied species. Moreover, the presence of these accessory cells may have ecological significance, as they accelerate the egg's production through trophic support of the growing oocyte.

The freshwater sponge, Ephydatia muelleri , is an emerging model system for studying animal:microbe symbioses. Intracellular green microalgae are one of the more common symbionts that live in a facultative mutualism with E. muelleri . While these symbioses have long been known, the identity of the algal symbionts in E. muelleri cells has not been studied in detail. Here, we isolate and characterize endosymbiotic algae from E. muelleri collected from different geographic locations. We find that the algae can be transmitted through asexually produced gemmules and importantly that they can form symbioses with different, differentiated sponge cell types in the adult sponge. Our findings indicate that at least two algal lineages form endosymbioses with E. muelleri . One of the lineages includes species commonly found in samples from two locations in Canada and one in the United States (clade 1: closely related to Auxenochlorella pyrenoidosa ). The other clade includes algae found in sponges from one site in Maine, USA, and Lewiniosphaera symbiontica , which is a strain isolated in 1956 from the freshwater sponge Spongilla . We compared microbiomes found in cultures of microalgae as well as the original sponge hosts, and found that very similar bacterial microbiomes associate with both clades (91 orders of Bacteria are shared among the samples we compared). The microbiomes found in the cultures resemble, with a high degree of overlap, the microbiome associated with the sponge host.

Well-annotated and contiguous genomes are an indispensable resource for understanding the evolution, development, and metabolic capacities of organisms. Sponges, an ecologically important non-bilaterian group of primarily filter-feeding sessile aquatic organisms, are underrepresented with respect to available genomic resources. Here we provide a high-quality and well-annotated genome of Aphrocallistes vastus, a glass sponge (Porifera: Hexactinellida) that forms large reef structures off the coast of British Columbia (Canada). We show that its genome is approximately 80 Mb, small compared to most other metazoans, and contains nearly 2500 nested genes, more than other genomes. Hexactinellida is characterized by a unique skeletal architecture made of amorphous silicon dioxide (SiO2), and we identified 419 differentially expressed genes between the osculum, i.e. the vertical growth zone of the sponge, and the main body. Among the upregulated ones, mineralization-related genes such as glassin, as well as collagens and actins, dominate the expression profile during growth. Silicateins, suggested being involved in silica mineralization, especially in demosponges, were not found at all in the A. vastus genome and suggests that the underlying mechanisms of SiO2 deposition in the Silicea sensu stricto (Hexactinellida + Demospongiae) may not be homologous.

BackgroundExplaining the emergence of the hallmarks of bilaterians is a central focus of evolutionary developmental biology-evodevo-and evolutionary genomics. For this purpose, we must both expand and also refine our knowledge of non-bilaterian genomes, especially by studying early branching animals, in particular those in the metazoan phylum Porifera.ResultsWe present a comprehensive analysis of the first whole genome of a glass sponge, Oopsacas minuta, a member of the Hexactinellida. Studying this class of sponge is evolutionary relevant because it differs from the three other Porifera classes in terms of development, tissue organization, ecology, and physiology. Although O. minuta does not exhibit drastic body simplifications, its genome is among the smallest of animal genomes sequenced so far, and surprisingly lacks several metazoan core genes (including Wnt and several key transcription factors). Our study also provides the complete genome of a symbiotic Archaea dominating the associated microbial community: a new Thaumarchaeota species.ConclusionsThe genome of the glass sponge O. minuta differs from all other available sponge genomes by its compactness and smaller number of encoded proteins. The unexpected loss of numerous genes previously considered ancestral and pivotal for metazoan morphogenetic processes most likely reflects the peculiar syncytial tissue organization in this group. Our work further documents the importance of convergence during animal evolution, with multiple convergent evolution of septate-like junctions, electrical-signaling and multiciliated cells in metazoans.

Freshwater sponges produce overwintering cysts called gemmules that are full of stem cells, allowing them to survive harsh winter months. The gemmules of Ephydatia muelleri can be kept in 3°C for months, and in -80°C for years, while still remaining viable to hatch and develop into a functional sponge, as outlined in the protocol: "Hatching and freezing gemmules from the freshwater sponge Ephydatia muelleri" (https://dx.doi.org/10.17504/protocols.io.863hzgn). With a recently published chromosome-level assembly of the genome (Kenny et al., 2020), along with multiple transcriptomes, this makes Ephydatia muelleri an ideal sponge species to establish as a model sponge species that can be used in labs world-wide. While the gemmules used in the lab are usually collected from rivers and lakes every season for practical reasons, there have been studies showing the gemmulation process can be induced. A classic study looking at this process was conducted by Rasmont in 1974, who used theophylline to induce gemmulation in the freshwater sponge Ephydatia fluviatilis. Using up to 400uM of theophylline, this study showed that gemmulation can be induced in 100% of the sponges used in the experiments, and can occur as fast as 4 days after treatment with theophylline. While this is an interesting and important process to understand the biology of freshwater sponges, the ease and practicality of collecting masses of gemmules from wild populations most likely led to this process being rarely revisited in the lab. Here, we outline a protocol for inducing gemmulation in the freshwater sponge Ephydatia muelleri using theophylline, establishedby revisiting the study done by Rasmont (1974). In E. muelleri, the gemmulation process takes longer than in E. fluviatilis, with the fastest documented gemmulation detected at 8 days after treatment with theophylline, and taking up to 12+ days in some cases. The gemmulation process usually completes within 4-5 days once the gemmulation process can be detected, and results in a smaller gemmule size than the ones obtained from wild populations. The newly formed gemmules take roughly 6-7 weeks to mature in the 3°C incubator before they can be plated to hatch. While this is a lengthy process, being able to induce gemmulation in the lab increases the potential for using E. muelleri in transfection studies. References Kenny, N.J., Francis, W.R., Rivera-Vicéns, R.E. et al. Tracing animal genomic evolution with the chromosomal-level assembly of the freshwater sponge Ephydatia muelleri. Nature Communications. 2020. 11, 3676. https://doi.org/10.1038/s41467-020-17397-w. Rasmont R. Stimulation of cell aggregation by theophylline in the asexual reproduction of fresh-water sponges (Ephydatia fluviatilis). Experientia. 1974. Jul 15;30(7):792-4. doi: 10.1007/BF01924190. PMID: 4367998.

The timing of early animal evolution remains one of the biggest conundrums in biology. Molecular data suggest Porifera diverged from the metazoan lineage some 800 Ma to 650 Ma, which contrasts with the earliest widely accepted fossils of sponges at 535 Ma. However, the lack of criteria by which to recognize the earliest animals in the fossil record presents a challenge. The sponge body plan is unchanged since the early Cambrian, which makes a sponge-type animal a good candidate for the earliest fossils. Here we propose a method for identifying an organism as sponge grade by translating the sponge pump character into a quantifiable morphological trait. We show that the ratio between the two major components of the aquiferous system, the cross-sectional area of the osculum (OSA) and the surface area of the whole sponge (SA), is an effective metric of the pump character of extant sponges and that the slope of this ratio is distinct for three classes of Porifera: Demospongiae, Calcarea, and Hexactinellida. Furthermore, this metric is effective at distinguishing as sponges both extant taxa and fossils from two extremes of the Phanerozoic, the Cambrian and Paleogene. We tested this metric on the putative Ediacaran sponge Thectardis avalonensis from Mistaken Point, Newfoundland, and found Thectardis fits both with Cambrian sponges and with modern demosponges. These analyses show that the OSA/SA ratio is a reliable character by which to identify fossils as sponge grade, opening up exciting possibilities for classifying new fossils as sponges.

The freshwater sponge Ephydatia muelleri is an emerging sponge model system. This species has clonal asexual reproduction stages called gemmules that are long-lasting and easily maintained in the lab, and a chromosome-level assembly of the genome (Kenny et al., 2020) with molecular resources (ephybase.ca), making this an easily used species for study by researchers around the world. Sponges grow attached to substrates, and one technical difficulty when working with species are attached to a substrate, is how to transfer samples to and from different solutions and vessels for experimental manipulations. In the case of E. muelleri, transfer is usually done with gemmules before they hatch and attach, or with sponges that are attached to small coverslips. Here, we outline a simple and effective method to grow E. muelleri on 2% agarose, a substrate that allows them to be peeled off with little damage, and transfer to other containers at any stage. Once transferred and left alone, they re-attach readily. E. muelleri development from gemmules are referred to as stages: stage 1 is just prehatching; stage 2 is hatched with no aquiferous system; stage 3 has 'pockets' of water spaces that are growing and merging with each other; stage 4 has an incipient but not fully organized canal system; stage 5 has a fully developed canal system with osculum vent. Although all stages can be easily peeled off the agarose substrate, sponges at earlier stages (2-3) develop at the same rate as undisturbed sponges after transfer, allowing direct comparison with unmanipulated control samples. This method allows different stages of E. muelleri to be transferred into cuvettes for electroporation, or into a solution for pharmacological treatment, or onto a new substrate for imaging. We also describe the best mechanism of timing hatching of gemmules. Gemmules are 'dormant' when kept at 3°C. Once placed at room temperature they undergo development within the collagen capsule, that involves differentiation of different cell types prior to their emergence from the micropyle (opening), as described by Höhr in 1977. This development can be thought of as "priming", or readying them for hatching. 'Priming' gemmules to the same level of hatching readiness, allows more control over timing of experiments. Primed gemmules also appear to be more resilient to both physical and chemical treatments. The gemmules slowly get primed once cleaned, or thawed from -80°C. A general guideline to prime gemmules is: 1 week in a 3°C incubator = 1 less day it takes for the gemmules to hatch after plating 1 day at room temperature = 1 less day it takes for the gemmules to hatch after plating Once they are over-primed (usually after 4-5 weeks in the 3°C incubator), they will start hatching even when kept at 3°C. We suggest that for reproducibility of experiments that concern the stage of development, experiments using E. muelleri gemmules would benefit from using primed gemmules or indicating how primed the gemmules were. Reference Kenny, N.J., Francis, W.R., Rivera-Vicéns, R.E. et al. Tracing animal genomic evolution with the chromosomal-level assembly of the freshwater sponge Ephydatia muelleri. Nature Communications. 2020. 11, 3676. https://doi.org/10.1038/s41467-020-17397-w. Höhr, D. 1977. Differenzierungsvorgänge in der keimenden Gemmula von Ephydatia fluviatilis. Rouxs Arch. Dev. Biol. 182: 329-346.

Sponges are interesting animal models for regeneration studies, since even from dissociated cells, they are able to regenerate completely. In particular, explants are model systems that can be applied to many sponge species, since small fragments of sponges can regenerate all elements of the adult, including the oscula and the ability to pump water. The morphological aspects of regeneration in sponges are relatively well known, but the molecular machinery is only now starting to be elucidated for some sponge species. Here, we have used an explant system of the demosponge Halichondria panicea to understand the molecular machinery deployed during regeneration of the aquiferous system. We sequenced the transcriptomes of four replicates of the 5-day explant without an osculum (NOE), four replicates of the 17-18-day explant with a single osculum and pumping activity (PE) and also four replicates of field-collected individuals with regular pumping activity (PA), and performed differential gene expression analysis. We also described the morphology of NOE and PE samples using light and electron microscopy. Our results showed a highly disorganised mesohyl and disarranged aquiferous system in NOE that is coupled with upregulated pathways of ciliogenesis, organisation of the ECM, and cell proliferation and survival. Once the osculum is formed, genes involved in "response to stimulus in other organisms" were upregulated. Interestingly, the main molecular machinery of vasculogenesis described in vertebrates was activated during the regeneration of the aquiferous system. Notably, vasculogenesis markers were upregulated when the tissue was disorganised and about to start forming canals (NOE) and angiogenic stimulators and ECM remodelling machineries were differentially expressed once the aquiferous system was in place (PE and PA). Our results are fundamental to better understanding the molecular mechanisms involved in the formation of the aquiferous system in sponges, and its similarities with the early onset of blood-vessel formation in animal evolution.

Sponges, among the oldest extant multicellular organisms on Earth,1 play a key role in the cycling of nutrients in many aquatic ecosystems.2-5 They need to employ strategies to prevent clogging of their internal filter system by solid wastes,6-8 but self-cleaning mechanisms are largely unknown. It is commonly assumed that sponges remove solid waste with the outflowing water through distinct outflow openings (oscula).3,9 Here, we present time-lapse video footage and analyses of sponge waste revealing a completely different mechanism of particle removal in the Caribbean tube sponge Aplysina archeri. This sponge actively moves particle-trapping mucus against the direction of its internal water flow and ejects it into the surrounding water from its seawater inlet pores (ostia) through periodic surface contractions that have been described earlier as "sneezing."10,11 Visually, it appears as if the sponge is continuously streaming mucus-embedded particles and sneezes to shed this particulate waste, resulting in a notable flux of detritus that is actively consumed by sponge-associated fauna. The new data are used to estimate production of detritus for this abundant sponge on Caribbean coral reefs. Last, we discuss why waste removal from the sponge inhalant pores may be a common feature among sponges and compare the process in sponges to equivalent mechanisms of mucus transport in other animals, including humans.

Trichoplax adhaerens is an enigmatic animal with an extraordinarily simple morphology and a cellular organization, which are the focus of current research. Protocols outlined here provide detailed descriptions of advanced techniques for light and electron microscopic studies of Trichoplax. Studies using these techniques have enhanced our understanding of cell type diversity and function in placozoans and have provided insight into the evolution, development, and physiology of this little understood group.