Body-Plan Reorganization in a Sponge Correlates with Microbiome Change.

Mounting evidence suggests that animals and their associated bacteria interact via intricate molecular mechanisms, and it is hypothesized that disturbances to the microbiome influence animal development. Here, we show that the loss of a key photosymbiont (i.e., bleaching) upon shading correlates with a stark body-plan reorganization in the common aquarium cyanosponge Lendenfeldia chondrodes. The morphological changes observed in shaded sponges include the development of a thread-like morphology that contrasts with the flattened, foliose morphology of control specimens. The microanatomy of shaded sponges markedly differed from that of control sponges, with shaded specimens lacking a well-developed cortex and choanosome. Also, the palisade of polyvacuolar gland-like cells typical in control specimens was absent in shaded sponges. The morphological changes observed in shaded specimens are coupled with broad transcriptomic changes and include the modulation of signaling pathways involved in animal morphogenesis and immune response, such as the Wnt, transforming growth factor ß (TGF-ß), and TLR-ILR pathways. This study provides a genetic, physiological, and morphological assessment of the effect of microbiome changes on sponge postembryonic development and homeostasis. The correlated response of the sponge host to the collapse of the population of symbiotic cyanobacteria provides evidence for a coupling between the sponge transcriptomic state and the state of its microbiome. This coupling suggests that the ability of animals to interact with their microbiomes and respond to microbiome perturbations has deep evolutionary origins in this group.

Research priorities for the sustainability of coral-rich western Pacific seascapes.

Nearly a billion people depend on tropical seascapes. The need to ensure sustainable use of these vital areas is recognised, as one of 17 policy commitments made by world leaders, in Sustainable Development Goal (SDG) 14 ('Life below Water') of the United Nations. SDG 14 seeks to secure marine sustainability by 2030. In a time of increasing social-ecological unpredictability and risk, scientists and policymakers working towards SDG 14 in the Asia-Pacific region need to know: (1) How are seascapes changing? (2) What can global society do about these changes? and (3) How can science and society together achieve sustainable seascape futures? Through a horizon scan, we identified nine emerging research priorities that clarify potential research contributions to marine sustainability in locations with high coral reef abundance. They include research on seascape geological and biological evolution and adaptation; elucidating drivers and mechanisms of change; understanding how seascape functions and services are produced, and how people depend on them; costs, benefits, and trade-offs to people in changing seascapes; improving seascape technologies and practices; learning to govern and manage seascapes for all; sustainable use, justice, and human well-being; bridging communities and epistemologies for innovative, equitable, and scale-crossing solutions; and informing resilient seascape futures through modelling and synthesis. Researchers can contribute to the sustainability of tropical seascapes by co-developing transdisciplinary understandings of people and ecosystems, emphasising the importance of equity and justice, and improving knowledge of key cross-scale and cross-level processes, feedbacks, and thresholds.

Digital Marine: An online platform for blended learning in a marine experimental biology module, the Schmid Training Course.

For over 20 years, the Schmid Training Course (STC) has offered unique opportunities for marine biology students from European universities to learn about marine model organisms. While the topics of the course have continuously changed over the years with the advent of new research techniques and discoveries, the pedagogical approach has remained largely the same - a combination of lectures, lab practicals, and field excursions. Several life science researchers, who have taught in the STC for many years, sought to bring the course's pedagogical approach into the 21st century, and with the support of Erasmus+ Programme of the European Community funding, the Digital Marine project was developed. Digital Marine began in 2018 as an international partnership between the six research centers from which the STC instructors hail, and its main objective was to introduce a flipped, blended approach to learning and teaching with respect to established and emerging marine biological model systems. The Digital Marine platform, which covers 12 marine model organisms, is now publicly available.

Hybrid Genome Assemblies of Marine Bacteria Isolated from the Sponge Sycon capricorn.

Sponges have complex relationships with bacteria, the roles of which include food, important components of the holobiont, pathogens, and accidentally accumulated elements of the environment. Consequently, sponges are reservoirs of microbial genomes and novel compounds. Therefore, we isolated and sequenced the whole genomes of bacterial species from the calcareous sponge Sycon capricorn.

Molecular techniques and their limitations shape our view of the holobiont.

It is now recognised that the biology of almost any organism cannot be fully understood without recognising the existence and potential functional importance of associated microbes. Arguably, the emergence of this holistic viewpoint may never have occurred without the development of a crucial molecular technique, 16S rDNA amplicon sequencing, which allowed microbial communities to be easily profiled across a broad range of contexts. A diverse array of molecular techniques are now used to profile microbial communities, infer their evolutionary histories, visualise them in host tissues, and measure their molecular activity. In this review, we examine each of these categories of measurement and inference with a focus on the questions they make tractable, and the degree to which their capabilities and limitations shape our view of the holobiont.

Convergent evolution of a vertebrate-like methylome in a marine sponge.

Vertebrates have highly methylated genomes at CpG positions, whereas invertebrates have sparsely methylated genomes. This increase in methylation content is considered a major regulatory innovation of vertebrate genomes. However, here we report that a sponge, proposed as the potential sister group to the rest of animals, has a highly methylated genome. Despite major differences in genome size and architecture, we find similarities between the independent acquisitions of the hypermethylated state. Both lineages show genome-wide CpG depletion, conserved strong transcription factor methyl-sensitivity and developmental methylation dynamics at 5-hydroxymethylcytosine enriched regions. Together, our findings trace back patterns associated with DNA methylation in vertebrates to the early steps of animal evolution. Thus, the sponge methylome challenges previous hypotheses concerning the uniqueness of vertebrate genome hypermethylation and its implications for regulatory complexity.

The Role of Homology and Orthology in the Phylogenomic Analysis of Metazoan Gene Content.

Resolving the relationships of animals (Metazoa) is crucial to our understanding of the origin of key traits such as muscles, guts, and nerves. However, a broadly accepted metazoan consensus phylogeny has yet to emerge. In part, this is because the genomes of deeply diverging and fast-evolving lineages may undergo significant gene turnover, reducing the number of orthologs shared with related phyla. This can limit the usefulness of traditional phylogenetic methods that rely on alignments of orthologous sequences. Phylogenetic analysis of gene content has the potential to circumvent this orthology requirement, with binary presence/absence of homologous gene families representing a source of phylogenetically informative characters. Applying binary substitution models to the gene content of 26 complete animal genomes, we demonstrate that patterns of gene conservation differ markedly depending on whether gene families are defined by orthology or homology, that is, whether paralogs are excluded or included. We conclude that the placement of some deeply diverging lineages may exceed the limit of resolution afforded by the current methods based on comparisons of orthologous protein sequences, and novel approaches are required to fully capture the evolutionary signal from genes within genomes.

Differentiation and Transdifferentiation of Sponge Cells.

Over 100 years of sponge biology research has demonstrated spectacular diversity of cell behaviors during embryonic development, metamorphosis and regeneration. The past two decades have allowed the first glimpses into molecular and cellular mechanisms of these processes. We have learned that while embryonic development of sponges utilizes a conserved set of developmental regulatory genes known from other animals, sponge cell differentiation appears unusually labile. During normal development, and especially as a response to injury, sponge cells appear to have an uncanny ability to transdifferentiate. Here, I argue that sponge cell differentiation plasticity does not preclude homology of cell types and processes between sponges and other animals. Instead, it does provide a wonderful opportunity to better understand transdifferentiation processes in all animals.

Spicule formation in calcareous sponges: Coordinated expression of biomineralization genes and spicule-type specific genes.

The ability to form mineral structures under biological control is widespread among animals. In several species, specific proteins have been shown to be involved in biomineralization, but it is uncertain how they influence the shape of the growing biomineral and the resulting skeleton. Calcareous sponges are the only sponges that form calcitic spicules, which, based on the number of rays (actines) are distinguished in diactines, triactines and tetractines. Each actine is formed by only two cells, called sclerocytes. Little is known about biomineralization proteins in calcareous sponges, other than that specific carbonic anhydrases (CAs) have been identified, and that uncharacterized Asx-rich proteins have been isolated from calcitic spicules. By RNA-Seq and RNA in situ hybridization (ISH), we identified five additional biomineralization genes in Sycon ciliatum: two bicarbonate transporters (BCTs) and three Asx-rich extracellular matrix proteins (ARPs). We show that these biomineralization genes are expressed in a coordinated pattern during spicule formation. Furthermore, two of the ARPs are spicule-type specific for triactines and tetractines (ARP1 or SciTriactinin) or diactines (ARP2 or SciDiactinin). Our results suggest that spicule formation is controlled by defined temporal and spatial expression of spicule-type specific sets of biomineralization genes.

An ancient role for nitric oxide in regulating the animal pelagobenthic life cycle: evidence from a marine sponge.

In many marine invertebrates, larval metamorphosis is induced by environmental cues that activate sensory receptors and signalling pathways. Nitric oxide (NO) is a gaseous signalling molecule that regulates metamorphosis in diverse bilaterians. In most cases NO inhibits or represses this process, although it functions as an activator in some species. Here we demonstrate that NO positively regulates metamorphosis in the poriferan Amphimedon queenslandica. High rates of A. queenslandica metamorphosis normally induced by a coralline alga are inhibited by an inhibitor of nitric oxide synthase (NOS) and by a NO scavenger. Consistent with this, an artificial donor of NO induces metamorphosis even in the absence of the alga. Inhibition of the ERK signalling pathway prevents metamorphosis in concert with, or downstream of, NO signalling; a NO donor cannot override the ERK inhibitor. NOS gene expression is activated late in embryogenesis and in larvae, and is enriched in specific epithelial and subepithelial cell types, including a putative sensory cell, the globular cell; DAF-FM staining supports these cells being primary sources of NO. Together, these results are consistent with NO playing an activating role in induction of A. queenslandica metamorphosis, evidence of its highly conserved regulatory role in metamorphosis throughout the Metazoa.

Conservation and divergence of bHLH genes in the calcisponge Sycon ciliatum.

BACKGROUND: Basic Helix-Loop-Helix (bHLH) genes encode a large family of eukaryotic transcription factors, categorized into six high-order groups: pan-eukaryotic group B involved in regulation of cell cycle, metabolism, and development; holozoan-specific groups C and F involved in development and maintenance of homeostasis; and metazoan-specific groups A, D and E including well-studied genes, such as Atonal, Twist and Hairy, with diverse developmental roles including control of morphogenesis and specification of neurons. Current scenarios of bHLH evolution in animals are mainly based on the bHLH gene set found in the genome of demosponge Amphimedon queenslandica. In this species, the majority of the 21 identified bHLH genes belong to group B, and the single group A gene is orthologous to several neurogenic bilaterian subfamilies, including atonal and neurogenin. RESULTS: Given recently discovered differences in developmental toolkit components between siliceous and calcareous sponges, we have carried out genome-wide analysis of bHLH genes in Sycon ciliatum, an emerging calcisponge model. We identified 30 bHLH genes in this species, representing 12 individual families, including four group A families not found in Amphimedon, and two larger family groupings. Notably, the families represented in Sycon are only partially overlapping with those represented in Amphimedon. Developmental expression analysis of a subset of the identified genes revealed patterns consistent with deeply conserved roles, such as specification of sensory cells by Atona-related and stem cells by Myc genes. CONCLUSIONS: Our results demonstrate independent gene loss events in demosponges and calcisponges, implying a complex bHLH toolkit in the last common metazoan ancestor.

Sponges as models to study emergence of complex animals.

The emergence of complex animal life forms remains poorly understood despite substantial interest and research in this area. To be informative, the ideal models to study transitions from single-cell organisms to the first animals and then to mammalian-level complexity should be phylogenetically strategically placed and retain ancestral characters. Sponges (Porifera) are likely to be the earliest branching animal phylum. When analysed from morphological, genomic and developmental perspectives, sponges appear to combine features of single-cell eukaryotic organisms and the complex multicellular animals (Eumetazoa). Intriguingly, homologues of components of the eumetazoan regulatory networks specifying the endoderm, the germ-cells and stem cells and (neuro) sensory cells are expressed in sponge choanocytes, archaeocytes and larval sensory cells. Studies using sponges as model systems are already bringing insights into animal evolution, and have opened avenues to further research benefitting from the recent spectacular expansion of genomic technologies.

Surprisingly rich repertoire of Wnt genes in the demosponge Halisarca dujardini.

BACKGROUND: Wnt proteins are secreted signalling molecules found in all animal phyla. In bilaterian animals, including humans, Wnt proteins play key roles in development, maintenance of homeostasis and regeneration. While Wnt gene repertoires and roles are strongly conserved between cnidarians and bilaterians, Wnt genes from basal metazoans (sponges, ctenophores, placozoans) are difficult or impossible to assign to the bilaterian + cnidarian orthologous groups. Moreover, dramatic differences in Wnt numbers among basal metazoan exist, with only three present in the genome of Amphimedon queenslandica, a demosponge, and 21 in the genome of Sycon ciliatum, a calcisponge. To gain insight into the ancestral Wnt repertoire and function, we have chosen to investigate Wnt genes in Halisarca dujardini, a demosponge with relatively well described development and regeneration, and a very distant phylogenetic relationship to Amphimedon. RESULTS: Here we describe generation of a eukaryotic contamination-free transcriptome of Halisarca dujardini, and analysis of Wnt genes repertoire and expression in this species. We have identified ten Wnt genes, with only one orthologous to Amphimedon Wnt, and six appearing to be a result of a lineage specific expansion. Expression analysis carried out by in situ hybridization of adults and larvae revealed that two Halisarca Wnts are expressed in nested domains in the posterior half of the larvae, and six along the adult body axis, with two specific to the osculum. Strikingly, expression of one of the Wnt genes was elevated in the region undergoing regeneration. CONCLUSIONS: Our results demonstrated that the three Poriferan lineages (Demospongiae, Calcarea and Homoloscleromorpha) are characterized by highly diverse Wnt gene repertoires which do not display higher similarity to each other than they do to the non-sponge (i.e. ctenophore, cnidarian and bilaterian) repertoires. This is in striking contrast to the uniform Wnt repertoires in Cnidarians and Bilaterians, suggesting that the Wnt family composition became "fixed" only in the last common ancestor of Cnidarians and Bilaterians. In contrast, expression of Wnt genes in the apical region of sponge adults and the posterior region of sponge larvae suggests conservation of the Wnt role in axial patterning across the animal kingdom.

Extensive Mitochondrial mRNA Editing and Unusual Mitochondrial Genome Organization in Calcaronean Sponges.

One of the unusual features of DNA-containing organelles in general and mitochondria in particular is the frequent occurrence of RNA editing [1]. The term "RNA editing" refers to a variety of mechanistically unrelated biochemical processes that alter RNA sequence during or after transcription [2]. The editing can be insertional, deletional, or substitutional and has been found in all major types of RNAs [3, 4]. Although mitochondrial mRNA editing is widespread in some eukaryotic lineages [5-7], it is rare in animals, with reported cases limited both in their scope and in phylogenetic distribution [8-11] (see also [12]). While analyzing genomic data from calcaronean sponges Sycon ciliatum and Leucosolenia complicata, we were perplexed by the lack of recognizable mitochondrial coding sequences. Comparison of genomic and transcriptomic data from these species revealed the presence of mitochondrial cryptogenes whose transcripts undergo extensive editing. This editing consisted of single or double uridylate (U) insertions in pre-existing short poly(U) tracts. Subsequent analysis revealed the presence of similar editing in Sycon coactum and the loss of editing in Petrobiona massiliana, a hypercalcified calcaronean sponge. In addition, mitochondrial genomes of at least some calcaronean sponges were found to have a highly unusual architecture, with nearly all genes located on individual and likely linear chromosomes. Phylogenetic analysis of mitochondrial coding sequences revealed accelerated rates of sequence evolution in this group. The latter observation presents a challenge for the mutational-hazard hypothesis [13], which posits that mRNA editing should not occur in lineages with an elevated mutation rate.

Regulatory RNA at the root of animals: dynamic expression of developmental lincRNAs in the calcisponge Sycon ciliatum.

Long non-coding RNAs (lncRNAs) play important regulatory roles during animal development, and it has been hypothesized that an RNA-based gene regulation was important for the evolution of developmental complexity in animals. However, most studies of lncRNA gene regulation have been performed using model animal species, and very little is known about this type of gene regulation in non-bilaterians. We have therefore analysed RNA-Seq data derived from a comprehensive set of embryogenesis stages in the calcareous sponge Sycon ciliatum and identified hundreds of developmentally expressed intergenic lncRNAs (lincRNAs) in this species. In situ hybridization of selected lincRNAs revealed dynamic spatial and temporal expression during embryonic development. More than 600 lincRNAs constitute integral parts of differentially expressed gene modules, which also contain known developmental regulatory genes, e.g. transcription factors and signalling molecules. This study provides insights into the non-coding gene repertoire of one of the earliest evolved animal lineages, and suggests that RNA-based gene regulation was probably present in the last common ancestor of animals.

Transdifferentiation is a driving force of regeneration in Halisarca dujardini (Demospongiae, Porifera).

The ability to regenerate is widespread in the animal kingdom, but the regenerative capacities and mechanisms vary widely. To understand the evolutionary history of the diverse regeneration mechanisms, the regeneration processes must be studied in early-evolved metazoans in addition to the traditional bilaterian and cnidarian models. For this purpose, we have combined several microscopy techniques to study mechanisms of regeneration in the demosponge Halisarca dujardini. The objectives of this work are to detect the cells and morphogenetic processes involved in Halisarca regeneration. We show that in Halisarca there are three main sources of the new exopinacoderm during regeneration: choanocytes, archaeocytes and (rarely) endopinacocytes. Here we show that epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) occur during Halisarca regeneration. EMT is the principal mechanism during the first stages of regeneration, soon after the injury. Epithelial cells from damaged and adjacent intact choanocyte chambers and aquiferous canals assume mesenchymal phenotype and migrate into the mesohyl. Together with archaeocytes, these cells form an undifferentiated cell mass beneath of wound, which we refer to as a blastema. After the blastema is formed, MET becomes the principal mechanism of regeneration. Altogether, we demonstrate that regeneration in demosponges involves a variety of processes utilized during regeneration in other animals (e.g., cell migration, dedifferentiation, blastema formation) and points to the particular importance of transdifferentiation in this process. Further studies will be needed to uncover the molecular mechanisms governing regeneration in sponges.

Comparative analyses of developmental transcription factor repertoires in sponges reveal unexpected complexity of the earliest animals.

Developmental transcription factors (DTFs) control development of animals by affecting expression of target genes, some of which are transcription factors themselves. In bilaterians and cnidarians, conserved DTFs are involved in homologous processes such as gastrulation or specification of neurons. The genome of Amphimedon queenslandica, the first sponge to be sequenced, revealed that only a fraction of these conserved DTF families are present in demosponges. This finding was in line with the view that morphological complexity in the animal lineage correlates with developmental toolkit complexity. However, as the phylum Porifera is very diverse, Amphimedon's genome may not be representative of all sponges. The recently sequenced genomes of calcareous sponges Sycon ciliatum and Leucosolenia complicata allowed investigations of DTFs in a sponge lineage evolutionarily distant from demosponges. Surprisingly, the phylogenetic analyses of identified DTFs revealed striking differences between the calcareous sponges and Amphimedon. As these differences appear to be a result of independent gene loss events in the two sponge lineages, the last common ancestor of sponges had to possess a much more diverse repertoire of DTFs than extant sponges. Developmental expression of sponge homologs of genes involved in specification of the Bilaterian endomesoderm and the neurosensory cells suggests that roles of many DTFs date back to the last common ancestor of all animals. Strikingly, even DTFs displaying apparent pan-metazoan conservation of sequence and function are not immune to being lost from individual species genomes. The quest for a comprehensive picture of the developmental toolkit in the last common metazoan ancestor is thus greatly benefitting from the increasing accessibility of sequencing, allowing comparisons of multiple genomes within each phylum.

Calcareous sponge genomes reveal complex evolution of α-carbonic anhydrases and two key biomineralization enzymes.

BACKGROUND: Calcium carbonate biominerals form often complex and beautiful skeletal elements, including coral exoskeletons and mollusc shells. Although the ability to generate these carbonate structures was apparently gained independently during animal evolution, it sometimes involves the same gene families. One of the best-studied of these gene families comprises the α- carbonic anhydrases (CAs), which catalyse the reversible transformation of CO2 to HCO3 - and fulfill many physiological functions. Among Porifera -the oldest animal phylum with the ability to produce skeletal elements- only the class of calcareous sponges can build calcitic spicules, which are the extracellular products of specialized cells, the sclerocytes. Little is known about the molecular mechanisms of their synthesis, but inhibition studies suggest an essential role of CAs. In order to gain insight into the evolution and function of CAs in biomineralization of a basal metazoan species, we determined the diversity and expression of CAs in the calcareous sponges Sycon ciliatum and Leucosolenia complicata by means of genomic screening, RNA-Seq and RNA in situ hybridization expression analysis. Active biomineralization was located with calcein-staining. RESULTS: We found that the CA repertoires of two calcareous sponge species are strikingly more complex than those of other sponges. By characterizing their expression patterns, we could link two CAs (one intracellular and one extracellular) to the process of calcite spicule formation in both studied species. The extracellular biomineralizing CAs seem to be of paralogous origin, a finding that advises caution against assuming functional conservation of biomineralizing genes based upon orthology assessment alone. Additionally, calcareous sponges possess acatalytic CAs related to human CAs X and XI, suggesting an ancient origin of these proteins. Phylogenetic analyses including CAs from genomes of all non-bilaterian phyla suggest multiple gene losses and duplications and presence of several CAs in the last common ancestor of metazoans. CONCLUSIONS: We identified two key biomineralization enzymes from the CA-family in calcareous sponges and propose their possible interaction in spicule formation. The complex evolutionary history of the CA family is driven by frequent gene diversification and losses. These evolutionary patterns likely facilitated the numerous events of independent recruitment of CAs into biomineralization within Metazoa.

Calcisponges have a ParaHox gene and dynamic expression of dispersed NK homeobox genes.

Sponges are simple animals with few cell types, but their genomes paradoxically contain a wide variety of developmental transcription factors, including homeobox genes belonging to the Antennapedia (ANTP) class, which in bilaterians encompass Hox, ParaHox and NK genes. In the genome of the demosponge Amphimedon queenslandica, no Hox or ParaHox genes are present, but NK genes are linked in a tight cluster similar to the NK clusters of bilaterians. It has been proposed that Hox and ParaHox genes originated from NK cluster genes after divergence of sponges from the lineage leading to cnidarians and bilaterians. On the other hand, synteny analysis lends support to the notion that the absence of Hox and ParaHox genes in Amphimedon is a result of secondary loss (the ghost locus hypothesis). Here we analysed complete suites of ANTP-class homeoboxes in two calcareous sponges, Sycon ciliatum and Leucosolenia complicata. Our phylogenetic analyses demonstrate that these calcisponges possess orthologues of bilaterian NK genes (Hex, Hmx and Msx), a varying number of additional NK genes and one ParaHox gene, Cdx. Despite the generation of scaffolds spanning multiple genes, we find no evidence of clustering of Sycon NK genes. All Sycon ANTP-class genes are developmentally expressed, with patterns suggesting their involvement in cell type specification in embryos and adults, metamorphosis and body plan patterning. These results demonstrate that ParaHox genes predate the origin of sponges, thus confirming the ghost locus hypothesis, and highlight the need to analyse the genomes of multiple sponge lineages to obtain a complete picture of the ancestral composition of the first animal genome.