Freshwater Sponge Ephydatia Muelleri Research Articles

Microbiome of the freshwater sponge Ephydatia muelleri shares compositional and functional similarities with those of marine sponges.

Sponges are known for hosting diverse communities of microbial symbionts, but despite persistent interest in the sponge microbiome, most research has targeted marine sponges; freshwater sponges have been the focus of less than a dozen studies. Here, we used 16 S rRNA gene amplicon sequencing and shotgun metagenomics to characterize the microbiome of the freshwater sponge Ephydatia muelleri and identify potential indicators of sponge-microbe mutualism. Using samples collected from the Sooke, Nanaimo, and Cowichan Rivers on Vancouver Island, British Columbia, we show that the E. muelleri microbiome is distinct from the ambient water and adjacent biofilms and is dominated by Sediminibacterium, Comamonas, and unclassified Rhodospirillales. We also observed phylotype-level differences in sponge microbiome taxonomic composition among different rivers. These differences were not reflected in the ambient water, suggesting that other environmental or host-specific factors may drive the observed geographic variation. Shotgun metagenomes and metagenome-assembled genomes further revealed that freshwater sponge-associated bacteria share many genomic similarities with marine sponge microbiota, including an abundance of defense-related proteins (CRISPR, restriction-modification systems, and transposases) and genes for vitamin B12 production. Overall, our results provide foundational information on the composition and function of freshwater sponge-associated microbes, which represent an important yet underappreciated component of the global sponge microbiome.

Establishment of Host-Algal Endosymbioses: Genetic Response to Symbiont Versus Prey in a Sponge Host.

The freshwater sponge Ephydatia muelleri and its Chlorella-like algal partner is an emerging model for studying animal: algal endosymbiosis. The sponge host is a tractable laboratory organism, and the symbiotic algae are easily cultured. We took advantage of these traits to interrogate questions about mechanisms that govern the establishment of durable intracellular partnerships between hosts and symbionts in facultative symbioses. We modified a classical experimental approach to discern the phagocytotic mechanisms that might be co-opted to permit persistent infections, and identified genes differentially expressed in sponges early in the establishment of endosymbiosis. We exposed algal-free E. muelleri to live native algal symbionts and potential food items (bacteria and native heat-killed algae), and performed RNA-Seq to compare patterns of gene expression among treatments. We found a relatively small but interesting suite of genes that are differentially expressed in the host exposed to live algal symbionts, and a larger number of genes triggered by host exposure to heat-killed algae. The upregulated genes in sponges exposed to live algal symbionts were mostly involved in endocytosis, ion transport, metabolic processes, vesicle-mediated transport, and oxidation–reduction. One of the host genes, an ATP-Binding Cassette transporter that is downregulated in response to live algal symbionts, was further evaluated for its possible role in the establishment of the symbiosis. We discuss the gene expression profiles associated with host responses to living algal cells in the context of conditions necessary for long-term residency within host cells by phototrophic symbionts as well as the genetic responses to sponge phagocytosis and immune-driven pathways.

Cloning and expression of ATP N-glycosidase from the freshwater sponge Ephydatia muelleri

Previously we had discovered unusual enzymatic activity in the marine sponge Axinella polypoides, ATP N-glycosidase (Reintamm et al., 2003). We show here that the Ephydatia muelleri mRNA encoding protein with PNP_UDP_1 (phosphorylase superfamily) signature is the secreted ATP N-glycosidase. The functionality of the protein was established by recombinant expression in Pichia pastoris. In addition to the enzymatic domain, the full-length protein contains the N-terminal cysteine-rich domain belonging to the subfamily SCP_HrTT-1 (cd05559) of the SCP (sperm coating protein) superfamily (cl00133).

Deoxynucleoside 5-monophosphate N-glycosidase from a phylogenetically distant metazoa, sponge

Deoxynucleoside 5-monophosphate N-glycosidase or DNPH1 (former name Rcl) is a nucleotide hydrolase whose expression in mammalian cancer tissues has been associated with its tumorigenic potential. Therefore, the enzyme has been studied principally in rat and human models. We found the corresponding gene also in the freshwater sponge Ephydatia muelleri, an animal phylogenetically very distant from mammals. Here we report the expression and characterization of the recombinant DNPH1 from E. muelleri. The ancient homolog of mammalian enzyme in a sponge showed the substrate specificity and catalytic efficiency similar to that in higher animals. E. muelleri DNPH1 is inhibited by the purine nucleotides with different numbers of 5′-phosphate groups (n = 1–4). Our results demonstrate that GTP but also dGTP are the best inhibitors, followed by all other purine nucleotides that were tested. Hence, the functioning of DNPH1 in cells where the natural ATP and GTP concentrations are much higher than those of the substrates, dNMPs, should normally be downregulated. We demonstrate for the first time the existence of biologically relevant natural inhibitors of DNPH1, namely ATP and GTP.

Small inverted repeats drive mitochondrial genome evolution in Lake Baikal sponges

Demosponges, the largest and most diverse class in the phylum Porifera, possess mitochondrial DNA (mtDNA) markedly different from that in other animals. Although several studies investigated evolution of demosponge mtDNA among major lineages of the group, the changes within these groups remain largely unexplored. Recently we determined mitochondrial genomic sequence of the Lake Baikal sponge Lubomirskia baicalensis and described proliferation of small inverted repeats (hairpins) that occurred in it since the divergence between L. baicalensis and the most closely related cosmopolitan freshwater sponge Ephydatia muelleri. Here we report mitochondrial genomes of three additional species of Lake Baikal sponges: Swartschewskia papyracea, Rezinkovia echinata and Baikalospongia intermedia morpha profundalis (Demospongiae, Haplosclerida, Lubomirskiidae) and from a more distantly related freshwater sponge Corvomeyenia sp. (Demospongiae, Haplosclerida, Metaniidae). We use these additional sequences to explore mtDNA evolution in Baikalian sponges, paying particular attention to the variation in the rates of nucleotide substitutions and the distribution of hairpins, abundant in these genomes. We show that most of the changes in Lubomirskiidae mitochondrial genomes are due to insertion/deletion/duplication of these elements rather than single nucleotide substitutions. Thus inverted repeats can act as an important force in evolution of mitochondrial genome architecture and be a valuable marker for population- and species-level studies in this group. In addition, we infer (((Rezinkovia+Lubomirskia)+Swartschewskia)+Baikalospongia) phylogeny for the family Lubomirskiidae based on the analysis of mitochondrial coding sequences from freshwater sponges.

RNA interference in marine and freshwater sponges: actin knockdown in Tethya wilhelma and Ephydatia muelleriby ingested dsRNA expressing bacteria

BackgroundThe marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available.ResultsWe show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from 'knocking down' expression of the actin gene.ConclusionThis technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals.

RNA interference in marine and freshwater sponges

Background: The marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available. Results: We show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from ‘knocking down’ expression of the actin gene. Conclusion: This technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals.

Rapid Proliferation of Repetitive Palindromic Elements in mtDNA of the Endemic Baikalian Sponge Lubomirskia baicalensis

Animal mitochondrial DNA (mtDNA) is a remarkably compact molecule largely because of the scarcity of noncoding "selfish" DNA. Recently, however, we found that mitochondrial genomes of several phylogenetically diverse species of demosponges contain small repetitive palindromic sequences, interspersed within intergenic regions and fused in protein and ribosomal RNA genes. Here, I report and analyze the proliferation of such elements in the mitochondrial genome of the endemic sponge of Lake Baikal Lubomirskia baicalensis. Because Baikal sponges are closely related to the circumglobally distributed freshwater sponge Ephydatia muelleri with which they shared a common ancestor approximately 3-10 Ma, both the rate of single nucleotide substitutions and the rate of palindromic repeat insertions can be calculated in this system. I found the rate of nucleotide substitutions in mtDNA of freshwater sponges to be extremely low (0.5-1.6 x 10(-9) per site per year), more similar to that in plants than bilaterian animals. By contrast, the per/nucleotide rate of insertions of repetitive elements is at least four times higher. This rapid rate of proliferation combined with the broad phylogenetic distribution of hairpin elements can make them a defining force in the evolution of mitochondrial genomes of demosponges.

Grazing impact and phenology of the freshwater sponge Ephydatia muelleri and the bryozoans Plumatella emarginata and Fredericella sultana under experimental warming

Summary1. Both the clearance rates (CR) and abundances of the freshwater sponge Ephydatia muelleri and the bryozoans Plumatella emarginata and Fredericella sultana were investigated from autumn to spring under different temperature regimes. The experiments were performed in bypass channels of the River Rhine (Cologne, Germany) in which temperature could be manipulated.2. The impact of temperature increase on CRs depends upon the grazer: E. muelleri showed a clear increase in CRs with increasing temperature whereas P. emarginata was not significantly affected by experimental warming.3. Distinct differences in food preference were found for the sponge (which is an efficient grazer of bacteria and small algae) and for the bryozoan P. emarginata (which feeds primarily on large algae, and with no significant grazing on bacteria).4. In contrast to their temperature‐related patterns in CR, respiration of both P. emarginata and E. muelleri increased with temperature between 19 and 32 °C, suggesting that the risk of experiencing energy deficiency at high temperatures due to increased metabolic rates is particularly high for the bryozoan.5. A temperature elevation of 3 °C above the natural Rhine temperature resulted in a delay in the disappearance of active tissue and formation of resting stages for E. muelleri in autumn. This delay ranged from 8 (beginning of gemmulation) to 22 days (termination of gemmulation). In contrast, there was no distinct effect of warming on the disappearance of active zooids of the two bryozoan species in autumn. However, warming can positively affect the maintenance of active zooids during winter in F. sultana. In spring, the appearance of active zooids of P. emarginata was clearly stimulated by temperature elevations, whereas the hatching of both F. sultana and E. muelleri was hardly affected by warming.6. The study demonstrated different patterns in the thermal ecology of the two freshwater bryozoans and the sponge in comparison to other filter feeders, particularly mussels. Such patterns need to be considered when predicting the impact of temperature on pelagic‐benthic coupling in aquatic habitats.

Coordinated contractions effectively expel water from the aquiferous system of a freshwater sponge

In response to mechanical stimuli the freshwater sponge Ephydatia muelleri (Demospongiae, Haplosclerida, Spongillidae) carries out a series of peristaltic-like contractions that is effective in expelling clumps of waste material from the aquiferous system. Rates of contraction depend on the region of tissue they are propagating through: 0.3-1 microm s(-1) in the peripheral canals, 1-4 microm s(-1) in central canals, and 6-122 microm s(-1) in the osculum. Faster events include twitches of the entire sponge choanosome and contraction of the sheet-like apical pinacoderm that forms the outer surface of the animal. Contraction events are temporally and spatially coordinated. Constriction of the tip of the osculum leads to dilation of excurrent canals; fields of ostia in the apical pinacoderm close in unison just prior to contraction of the choanosome, apical pinacoderm and osculum. Relaxation returns the osculum, canals and the apical pinacoderm to their normal state, and three such coordinated 'inflation-contraction' responses typically follow a single stimulus. Cells in the mesohyl arrest crawling as a wave of contraction passes, suggesting an extracellular signal may pass between cells. Bundles of actin filaments traverse endopinacocytes of the apical pinacoderm. Actin-dense plaques join actin bundles in adjacent pinacocytes to form continuous tracts spanning the whole sponge. The orchestrated and highly repeatable series of contractions illustrates that cellular sponges are capable of coordinated behavioural responses even in the absence of neurons and true muscle. Propagation of the events through the pinacocytes also illustrates the presence of a functional epithelium in cellular sponges. These results suggest that control over a hydrostatic skeleton evolved prior to the origin of nerves and true muscle.

Retinoic acid down-regulates the expression of EmH-3 homeobox-containing gene in the freshwater sponge Ephydatiamuelleri

The effects of retinoic acid (RA), a common morphogen and gene expression regulator in vertebrates, were studied in the freshwater sponge Ephydatia muelleri, both on morphogenesis and on the expression of EmH-3 homeobox-containing gene . At 0.3 μM, RA had no noticeable influence on sponge development, slightly up-regulating EmH-3 expression. In contrast, in sponges reared in 10, 8 μM and to a lesser extent 2 μM RA, there was a strong down-regulation of EmH-3 expression after hatching. This induced modifications in cell composition and morphology, greatly disturbing normal development. Archaeocytes kept the features found in newly hatched sponges while choanocytes and a functional aquiferous system were completely absent. The inhibition of morphogenesis and down-regulation of EmH-3 expression were reversible when sponges were no longer subjected to RA. After RA removal, EmH-3 expression returned to the high values found in untreated sponges, archaeocytes differentiated into choanocytes and sponges achieved a normal development. These results clearly show that, in freshwater sponges, the most primitive metazoan, RA may also act as a morphogen, regulating the expression of a homeobox-containing gene. They demonstrate that the expression of EmH-3 is necessary for the differentiation of archaeocytes into choanocytes and hence for the formation of a complete functional aquiferous system.

Temporal and spatial expression of EmH-3, a homeobox-containing gene isolated from the freshwater sponge Ephydatia muelleri

Homeoboxes have been particularly valuable in identifying genes involved in development. This prompted us to look for homeobox-containing genes in sponges, the most primitive metazoans, and to explore the potential role of these genes in their development. Using the reverse transcription polymerase reaction (RT-PCR), we analyzed the expression of EmH-3 homeobox-containing gene at different stages of development, and in different cell-type populations. The patterns of EmH-3 expression show that this gene is expressed differentially in the course of development and in a cell-type specific manner. The level of transcripts increases from undetectable levels in resting gemmules to higher levels at the moment of hatching and throughout the sponge’s life. EmH-3 is strongly expressed in the pluripotent archaeocytes, whether isolated from fully differentiated sponges (adult archaeocytes) or from HU-treated sponges (embryonic archaeocytes). Conversely, in differentiated cells such as pinacocytes and choanocytes, EmH-3 expression is very weak and similar to that found in the resting gemmules. On the other hand, another freshwater sponge homeobox-containing gene, prox1 from Ephydatia fluviatilis is expressed almost at the same level at all stages of development and in all the investigated cell populations. Together, these results suggest that EmH-3 plays a role in cell determination and/or differentiation. In particular EmH-3 would determine which archaeocytes will multiply and undergo differentiation and which ones will remain undifferentiated.

Origin of the metazoan bodyplan: characterization and functional testing of the promoter of the homeobox gene EmH-3 from the freshwater sponge Ephydatia muelleri in mouse 3T3 cells.

Porifera [sponges] represent the lowest metazoan phylum, probably already existing prior to the 'Cambrian explosion'. Based on amino acid sequences deduced from cDNAs that code for structural proteins, the monophyly of Metazoa was established. Now we analyzed for the first time a promoter of a sponge gene for its activity in a heterologous cell system from higher Metazoa. The promoter of the homeobox gene EmH-3 was cloned and sequenced from a genomic library of the freshwater sponge Ephydatia muelleri. For the determination of functional promoter activity, transient transfection experiments in mouse NIH 3T3 cells were performed; the promoter was fused with the luciferase reporter gene. The data revealed that a 401 nt long promoter fragment, comprising several binding elements for metazoan transcription factors, showed the highest activity, while the 175 bp long promoter segment, comprising solely the TATA- and Cap boxes, showed only 25% of that activity. This result demonstrates that the sponge promoter is activated by factors present in mammalian cells and supports the view that Porifera, together with the other metazoan phyla, are of monophyletic origin.

Inorganic polyphosphates in the developing freshwater sponge Ephydatia muelleri: Effect of stress by polluted waters

AbstractRelatively high amounts of inorganic polyphosphates (approximately 55 μg of polyphosphate/g of wet weight) were found in the freshwater sponge Ephydatia muelleri, particularly in the gemmules (260 μg/g). Here we report that the polyphosphate content of this sponge changes during development and in response to adverse environmental conditions. Germination and hatching of gemmules of E. muelleri is accompanied by a strong decrease (by 94% at day 2) in polyphosphate level and a rise in exopolyphosphatase activity. On the other hand, induction of gemmulogenesis by theophylline results in an increase (by 61%) in polyphosphate content of sponge tissue. An increase in polyphosphate content and a decrease in exopolyphosphatase activity also occur during tissue regression when hatched sponges are exposed to polluted water from river. Nonionic organic compounds extracted from this water were identified as contaminants causing a rise in polyphosphate content of E. muelleri. The results show that measurement of polyphosphate level may be a promising method to detect responses of the freshwater sponge to polluted waters.

Formation of spicules by sclerocytes from the freshwater sponge Ephydatia muelleri in short-term cultures in vitro.

Cells from the freshwater sponge Ephydatia muelleri were isolated by dissociating hatching gemmules. During the first 24 h the cells reaggregated, but the aggregates progressively disintegrated again to single cells, among which the spicule-forming sclerocytes were recognized. Such cultures were used to study spicule (megascleres) formation in vitro. The isolated sclerocytes formed the organic central axial filament onto which they deposited inorganic silicon. The size of the spicules (200 to 350 microns in length) as well as the rate of spicule formation (1 to 10 microns/h) under in vitro conditions were similar to the values measured in vivo. Immediately after completion of spicule formation, or even before, the sclerocyte could start formation of a new spicule; 5% of the cells were in the process of forming two spicules simultaneously. Cultivation of sclerocytes in the absence of silicon resulted in the formation of the axial filament only. We succeeded in maintaining the sclerocytes in a proliferating and spicule-forming state for up to 3 mo. These results demonstrate that the establishment of short-term cell cultures from E. muelleri is possible; however, future studies must be undertaken to identify the growth factors required for a permanent culture of sponge cells.