Molecular machineries of ciliogenesis, cell survival, and vasculogenesis are differentially expressed during regeneration in explants of the demosponge Halichondria panicea.

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.

The Molecular Machinery of Gametogenesis in Geodia Demosponges (Porifera): Evolutionary Origins of a Conserved Toolkit across Animals.

All animals are capable of undergoing gametogenesis. The ability of forming haploid cells from diploid cells through meiosis and recombination appeared early in eukaryotes, whereas further gamete differentiation is mostly a metazoan signature. Morphologically, the gametogenic process presents many similarities across animal taxa, but little is known about its conservation at the molecular level. Porifera are the earliest divergent animals and therefore are an ideal phylum to understand evolution of the gametogenic toolkits. Although sponge gametogenesis is well known at the histological level, the molecular toolkits for gamete production are largely unknown. Our goal was to identify the genes and their expression levels which regulate oogenesis and spermatogenesis in five gonochoristic and oviparous species of the genus Geodia, using both RNAseq and proteomic analyses. In the early stages of both female and male gametogenesis, genes involved in germ cell fate and cell-renewal were upregulated. Then, molecular signals involved in retinoic acid pathway could trigger the meiotic processes. During later stages of oogenesis, female sponges expressed genes involved in cell growth, vitellogenesis, and extracellular matrix reassembly, which are conserved elements of oocyte maturation in Metazoa. Likewise, in spermatogenesis, genes regulating the whole meiotic cycle, chromatin compaction, and flagellum axoneme formation, that are common across Metazoa were overexpressed in the sponges. Finally, molecular signals possibly related to sperm capacitation were identified during late stages of spermatogenesis for the first time in Porifera. In conclusion, the activated molecular toolkit during gametogenesis in sponges was remarkably similar to that deployed during gametogenesis in vertebrates.

Mitochondrial evolution in the Demospongiae (Porifera): Phylogeny, divergence time, and genome biology.

The sponge class Demospongiae is the most speciose and morphologically diverse in the phylum Porifera, and the species within it are vital components of a range of ecosystems worldwide. Despite their ubiquity, a number of recalcitrant problems still remain to be solved regarding their phylogenetic inter-relationships, the timing of their appearance, and their mitochondrial biology, the latter of which is only beginning to be investigated. Here we generated 14 new demosponge mitochondrial genomes which, alongside previously published mitochondrial resources, were used to address these issues. In addition to phylogenomic analysis, we have used syntenic data and analysis of coding regions to forge a framework for understanding the inter-relationships between Demospongiae sub-classes and orders. We have also leveraged our new resources to study the mitochondrial biology of these clades in terms of codon usage, optimisation and gene expression, to understand how these vital cellular components may have contributed to the success of the Porifera. Our results strongly support a sister relationship between Keratosa and (Verongimorpha + Heteroscleromorpha), contradicting previous studies using nuclear markers. Our study includes one species of Clionaida, and show for the first time support for a grouping of Suberitida+(Clionaida+(Tethyida + Poecilosclerida). The findings of our phylogenetic analyses are supported by in-depth examination of structural and coding-level evidence from our mitochondrial data. A time-calibrated phylogeny estimated the origin of Demospongiae in the Cambrian (~529 Mya), and suggests that most demosponge order crown-groups emerged in the Mesozoic. This work therefore provides a robust basis for considering demosponge phylogenetic relationships, as well as essential mitochondrial data for understanding the biological basis for their success and diversity.

Implications of population connectivity studies for the design of marine protected areas in the deep sea: An example of a demosponge from the Clarion-Clipperton Zone.

The abyssal demosponge Plenaster craigi inhabits the Clarion-Clipperton Zone (CCZ) in the northeast Pacific, a region with abundant seafloor polymetallic nodules with potential mining interest. Since P. craigi is a very abundant encrusting sponge on nodules, understanding its genetic diversity and connectivity could provide important insights into extinction risks and design of marine protected areas. Our main aim was to assess the effectiveness of the Area of Particular Environmental Interest 6 (APEI-6) as a potential genetic reservoir for three adjacent mining exploration contract areas (UK-1A, UK-1B and OMS-1A). As in many other sponges, COI showed extremely low variability even for samples ~900 km apart. Conversely, the 168 individuals of P. craigi, genotyped for 11 microsatellite markers, provided strong genetic structure at large geographical scales not explained by isolation by distance (IBD). Interestingly, we detected molecular affinities between samples from APEI-6 and UK-1A, despite being separated ~800 km. Although our migration analysis inferred very little progeny dispersal of individuals between areas, the major differentiation of OMS-1A from the other areas might be explained by the occurrence of predominantly northeasterly transport predicted by the HYCOM hydrodynamic model. Our study suggests that although APEI-6 does serve a conservation role, with species connectivity to the exploration areas, it is on its own inadequate as a propagule source for P. craigi for the entire eastern portion of the CCZ. Our new data suggest that an APEI located to the east and/or the south of the UK-1, OMS-1, BGR, TOML and NORI areas would be highly valuable.

Lipopolysaccharides from Commensal and Opportunistic Bacteria: Characterization and Response of the Immune System of the Host Sponge Suberites domuncula.

Marine sponges harbor a rich bacterioflora with which they maintain close relationships. However, the way these animals make the distinction between bacteria which are consumed to meet their metabolic needs and opportunistic and commensal bacteria which are hosted is not elucidated. Among the elements participating in this discrimination, bacterial cell wall components such as lipopolysaccharides (LPS) could play a role. In the present study, we investigated the LPS chemical structure of two bacteria associated with the sponge Suberites domuncula: a commensal Endozoicomonas sp. and an opportunistic Pseudoalteromonas sp. Electrophoretic patterns indicated different LPS structures for these bacteria. The immunomodulatory lipid A was isolated after mild acetic acid hydrolysis. The electrospray ionization ion-trap mass spectra revealed monophosphorylated molecules corresponding to tetra- and pentaacylated structures with common structural features between the two strains. Despite peculiar structural characteristics, none of these two LPS influenced the expression of the macrophage-expressed gene S. domuncula unlike the Escherichia coli ones. Further research will have to include a larger number of genes to understand how this animal can distinguish between LPS with resembling structures and discriminate between bacteria associated with it.

First insights into the Aurelia aurita transcriptome response upon manipulation of its microbiome.

Introduction: The associated diverse microbiome contributes to the overall fitness of Aurelia aurita, particularly to asexual reproduction. However, how A. aurita maintains this specific microbiome or reacts to manipulations is unknown. Methods: In this report, the response of A. aurita to manipulations of its native microbiome was studied by a transcriptomics approach. Microbiome-manipulated polyps were generated by antibiotic treatment and challenging polyps with a non-native, native, and potentially pathogenic bacterium. Total RNA extraction followed by RNAseq resulted in over 155 million reads used for a de novo assembly. Results: The transcriptome analysis showed that the antibiotic-induced change and resulting reduction of the microbiome significantly affected the host transcriptome, e.g., genes involved in processes related to immune response and defense mechanisms were highly upregulated. Similarly, manipulating the microbiome by challenging the polyp with a high load of bacteria (2 × 107 cells/polyp) resulted in induced transcription of apoptosis-, defense-, and immune response genes. A second focus was on host-derived quorum sensing interference as a potential defense strategy. Quorum Quenching (QQ) activities and the respective encoding QQ-ORFs of A. aurita were identified by functional screening a cDNA-based expression library generated in Escherichia coli. Corresponding sequences were identified in the transcriptome assembly. Moreover, gene expression analysis revealed differential expression of QQ genes depending on the treatment, strongly suggesting QQ as an additional defense strategy. Discussion: Overall, this study allows first insights into A. aurita's response to manipulating its microbiome, thus paving the way for an in-depth analysis of the basal immune system and additional fundamental defense strategies.

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.

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.

Trimitomics: An efficient pipeline for mitochondrial assembly from transcriptomic reads in nonmodel species.

Mitochondrial resources are of known utility to many fields of phylogenetic, population and molecular biology. Their combination of faster and slower-evolving regions and high copy number enables them to be used in many situations where other loci are unsuitable, with degraded samples and after recent speciation events.The advent of next-generation sequencing technologies (and notably the Illumina platform) has led to an explosion in the number of samples that can be studied at transcriptomic level, at relatively low cost. Here we describe a robust pipeline for the recovery of mitochondrial genomes from these RNA-sequencing resources. This pipeline can be used on sequencing of a variety of depths, and reliably recovers the protein coding and ribosomal gene complements of mitochondria from almost any transcriptomic sequencing experiment. The complete sequence of the mitochondrial genome can also be recovered when sequencing is performed in sufficient depth. We show the efficacy of our pipeline using data from eight nonmodel invertebrates of six disparate phyla. Interestingly, among our poriferan data, where microbiological symbionts are known empirically to make mitochondrial assembly difficult, this pipeline proved especially useful. Our pipeline will allow the recovery of mitochondrial data from a variety of previously sequenced samples, and add an additional angle of enquiry to future RNA-sequencing efforts, simplifying the process of mitochondrial genome assembly for even the most recalcitrant clades and adding these data to the scientific record for a range of future uses.

A de novo transcriptome assembly for the bath sponge Spongia officinalis, adjusting for microsymbionts.

OBJECTIVES: We report a transcriptome acquisition for the bath sponge Spongia officinalis, a non-model marine organism that hosts rich symbiotic microbial communities. To this end, a pipeline was developed to efficiently separate between bacterial expressed genes from those of eukaryotic origin. The transcriptome was produced to support the assessment of gene expression and, thus, the response of the sponge, to elevated temperatures, replicating conditions currently occurring in its native habitat. DATA DESCRIPTION: We describe the assembled transcriptome along with the bioinformatic pipeline used to discriminate between signals of metazoan and prokaryotic origin. The pipeline involves standard read pre-processing steps and incorporates extra analyses to identify and filter prokaryotic reads out of the analysis. The proposed pipeline can be followed to overcome the technical RNASeq problems characteristic for symbiont-rich metazoan organisms with low or non-existent tissue differentiation, such as sponges and cnidarians. At the same time, it can be valuable towards the development of approaches for parallel transcriptomic studies of symbiotic communities and the host.

Insights into the reproduction of some Antarctic dendroceratid, poecilosclerid, and haplosclerid demosponges.

Sponges are a dominant element of the Antarctic benthic communities, posing both high species richness and large population densities. Despite their importance in Antarctic ecosystems, very little is known about their reproductive patterns and strategies. In our study, we surveyed the tissue of six different species for reproductive elements, namely, Dendrilla antarctica Topsent, 1905 (order Dendroceratida), Phorbas areolatus (Thiele, 1905), Kirkpatrickia variolosa (Kirkpatrick, 1907), and Isodictya kerguelenensis (Ridley & Dendy, 1886) (order Poecilosclerida), and Hemigellius pilosus (Kirkpatrick, 1907) and Haliclona penicillata (Topsent, 1908) (Haplosclerida). Samples of these six species containing various reproductive elements were collected in Deception Island and were processed for both light and transmission electron microscopy (TEM). Even though we were not able to monitor the entire reproductive cycle, due to time and meteorological conditions, we report important aspects of the reproduction of these species. This includes oocyte and embryo morphology and cell ultrastructure, follicular structures and nurse cell activity, as well as vitellogenesis. All species were brooding their embryos within their mesohyl. Both oocytes and embryos were registered in the majority of the studied species, and a single sperm cell being carried to an egg for fertilization was observed in H. penicillata. While the reproductive periods of all species coincided temporally, some of them seemed to rely on a single spawning event, this being suggested by the synchronic oogenesis and embryogenesis occurrence of D. antarctica, P. areolatus and I. kerguelenensis. In contrast, K. variolosa had an asynchronous embryo development, which suggests several larval release events. Our results suggest that differences in the reproductive strategies and morphological traits might succeed in the coexistence of these species at the same habitat avoiding the direct competition between them.