Methylmercury exposure of the sponge O. lobularis induces strong tissue and cell defects.

Mediterranean marine biota suffers from various anthropogenic threats. Among them, pollutants such as mercury (Hg) represent important environmental issues that are exacerbated by bioaccumulation and bioamplification along food webs via its organic form, monomethylmercury (MMHg). To date, very little is known regarding the impact of mercury on Porifera and the few available studies have been exclusively focused on Demospongiae. This work studies the effect of MMHgCl at different biological levels of Oscarella lobularis (Porifera, Homoscleromorpha). Bioaccumulation assays show that MMHgCl significantly accumulated in sponge tissues after a 96-h exposure to 0.1 µg L-1. Toxicity assays (LC5096h) show a sensibility that depends on life-stage (adult vs bud). Additionally, we show that the exposure to 1 µg L-1 MMHgCl negatively impacts the epithelial integrity and the regeneration process in buds, as shown by the loss of cell-cell contacts and the alteration of osculum morphogenesis. For the first time in a sponge, a whole set of genes classically involved in metal detoxification and in antioxidant response were identified. Significant changes in catalase, superoxide dismutase and nuclear factor (erythroid-derived 2)-like 2 expressions in exposed juveniles were measured. Such an integrative approach from the physiological to the molecular scales on a non-model organism expands our knowledge concerning sensitivity and toxicity mechanisms induced by MMHg in Porifera, raising new questions regarding the possible defences used by marine sponges.

The compact genome of the sponge Oopsacas minuta (Hexactinellida) is lacking key metazoan core genes.

BACKGROUND: Explaining 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. RESULTS: We 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. CONCLUSIONS: The 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.

Evolution of mechanisms controlling epithelial morphogenesis across animals: new insights from dissociation-reaggregation experiments in the sponge Oscarella lobularis.

BACKGROUND: The ancestral presence of epithelia in Metazoa is no longer debated. Porifera seem to be one of the best candidates to be the sister group to all other Metazoa. This makes them a key taxon to explore cell-adhesion evolution on animals. For this reason, several transcriptomic, genomic, histological, physiological and biochemical studies focused on sponge epithelia. Nevertheless, the complete and precise protein composition of cell-cell junctions and mechanisms that regulate epithelial morphogenetic processes still remain at the center of attention. RESULTS: To get insights into the early evolution of epithelial morphogenesis, we focused on morphogenic characteristics of the homoscleromorph sponge Oscarella lobularis. Homoscleromorpha are a sponge class with a typical basement membrane and adhaerens-like junctions unknown in other sponge classes. We took advantage of the dynamic context provided by cell dissociation-reaggregation experiments to explore morphogenetic processes in epithelial cells in a non-bilaterian lineage by combining fluorescent and electron microscopy observations and RNA sequencing approaches at key time-points of the dissociation and reaggregation processes. CONCLUSIONS: Our results show that part of the molecular toolkit involved in the loss and restoration of epithelial features such as cell-cell and cell-matrix adhesion is conserved between Homoscleromorpha and Bilateria, suggesting their common role in the last common ancestor of animals. In addition, sponge-specific genes are differently expressed during the dissociation and reaggregation processes, calling for future functional characterization of these genes.

Staining and Tracking Methods for Studying Sponge Cell Dynamics.

To better understand the origin of animal cell types, body plans, and other morphological features, further biological knowledge and understanding are needed from non-bilaterian phyla, namely, Placozoa, Ctenophora, and Porifera. This chapter describes recent cell staining approaches that have been developed in three phylogenetically distinct sponge species-the homoscleromorph Oscarella lobularis, and the demosponges Amphimedon queenslandica and Lycopodina hypogea-to enable analyses of cell death, proliferation, and migration. These methods allow for a more detailed understanding of cellular behaviors and fates, and morphogenetic processes in poriferans, building on current knowledge of sponge cell biology that relies chiefly on classical (static) histological observations.

In Situ Hybridization Techniques in the Homoscleromorph Sponge Oscarella lobularis.

The Porifera are one of the best candidates as the sister group to all other metazoans. Studies on this phylum are therefore expected to shed light on the origin and early evolution of key animal features. Transcriptomic or genomic data acquired during the last 10 years have highlighted the conservation of most of the main genes and pathways involved in the development of the other metazoans. The next step is to determine how similar genetic tool boxes can result in widely dissimilar body plan organization, dynamics, and life histories. To answer these questions, three main axes of research are necessary: (1) conducting more gene expression studies; (2) developing knockdown protocols; and (3) reinterpreting sponge cell biology using modern tools. In this chapter we focus on the in situ hybridization (ISH) technique, needed to establish the spatiotemporal expression of genes, both on whole mount individuals and paraffin sections, and at different stages of development (adults, embryos, larvae, buds) of the homoscleromorph sponge Oscarella lobularis.

From seascape ecology to population genomics and back. Spatial and ecological differentiation among cryptic species of the red algae Lithophyllum stictiforme/L. cabiochiae, main bioconstructors of coralligenous habitats.

Ecosystem engineering species alter the physical structure of their environment and can create or modify habitats, having a massive impact on local biodiversity. Coralligenous reefs are highly diverse habitats endemic to the Mediterranean Sea built by calcareous benthic organisms among which Crustose Coralline Algae are the main engineering species. We analyzed the diversity of Lithophyllum stictiforme or L. cabiochiae in coralligenous habitats combining a multiple barcode and a population genomics approach with seascape features. Population genomics allowed disentangling pure spatial effects from environmental effects. We found that these taxa form a complex of eight highly divergent cryptic species that are easily identifiable using classic barcode markers (psbA, LSU, COI). Three factors have a significant effect on the relative abundances of these cryptic species: the location along the French Mediterranean coast, depth and Photosynthetic Active Radiation (PAR). The analysis of around 5000 SNPs for the most abundant species revealed genetic differentiation among localities in the Bay of Marseille but no differentiation between depths within locality. Thus, the effect of depth and PAR on cryptic species communities is not a consequence of restricted connectivity but rather due to differential settlement or survival among cryptic species. This differential is more likely driven by irradiance levels rather than by pressure or temperature. Both the genetic and species diversity patterns are congruent with the main patterns of currents in the Bay. Ecological differentiation among these engineering cryptic species, sensitive to ocean warming and acidification, could have important consequences on the diversity and structure of the coralligenous communities.

The Conservation of the Germline Multipotency Program, from Sponges to Vertebrates: A Stepping Stone to Understanding the Somatic and Germline Origins.

The germline definition in metazoans was first based on few bilaterian models. As a result, gene function interpretations were often based on phenotypes observed in those models and led to the definition of a set of genes, considered as specific of the germline, named the "germline core". However, some of these genes were shown to also be involved in somatic stem cells, thus leading to the notion of germline multipotency program (GMP). Because Porifera and Ctenophora are currently the best candidates to be the sister-group to all other animals, the comparative analysis of gene contents and functions between these phyla, Cnidaria and Bilateria is expected to provide clues on early animal evolution and on the links between somatic and germ lineages. Our present bioinformatic analyses at the metazoan scale show that a set of 18 GMP genes was already present in the last common ancestor of metazoans and indicate more precisely the evolution of some of them in the animal lineage. The expression patterns and levels of 11 of these genes in the homoscleromorph sponge Oscarella lobularis show that they are expressed throughout their life cycle, in pluri/multipotent progenitors, during gametogenesis, embryogenesis and during wound healing. This new study in a nonbilaterian species reinforces the hypothesis of an ancestral multipotency program.

Draft Genome Sequence of an Alphaproteobacterium Associated with the Mediterranean Sponge Oscarella lobularis.

While sequencing DNA purified from the homoscleromorph sponge Oscarella lobularis, we detected a large number of reads with strong similarity to available alphaproteobacteria gene sequences of family Rhodobacteraceae. Here, we present the genome sequence of this putative sponge symbiont that we propose to designate as "Candidatus Rhodobacter lobularis."

Mitochondrial Genome Sequence of the Glass Sponge Oopsacas minuta.

We report the complete mitochondrial genome sequence of the Mediterranean glass sponge Oopsacas minuta. This 19-kb mitochondrial genome has 24 noncoding genes (22 tRNAs and 2 rRNAs) and 14 protein-encoding genes coding for 11 subunits of respiratory chain complexes and 3 ATP synthase subunits.

Systematics and molecular phylogeny of the family oscarellidae (homoscleromorpha) with description of two new oscarella species.

The family Oscarellidae is one of the two families in the class Homoscleromorpha (phylum Porifera) and is characterized by the absence of a skeleton and the presence of a specific mitochondrial gene, tatC. This family currently encompasses sponges in two genera: Oscarella with 17 described species and Pseudocorticium with one described species. Although sponges in this group are relatively well-studied, phylogenetic relationships among members of Oscarellidae and the validity of genus Pseudocorticium remain open questions. Here we present a phylogenetic analysis of Oscarellidae using four markers (18S rDNA, 28S rDNA, atp6, tatC), and argue that it should become a mono-generic family, with Pseudocorticium being synonymized with Oscarella, and with the transfer of Pseudocorticium jarrei to Oscarella jarrei. We show that the genus Oscarella can be subdivided into four clades, each of which is supported by either a small number of morphological characters or by molecular synapomorphies. In addition, we describe two new species of Oscarella from Norwegian fjords: O. bergenensis sp. nov. and O. nicolae sp. nov., and we compare their morphology, anatomy, and cytology with other species in this genus. Internal anatomical characters are similar in both species, but details of external morphology and particularly of cytological characters provide diagnostic features. Our study also confirms that O. lobularis and O. tuberculata are two distinct polychromic sibling species. This study highlights the difficulties of species identification in skeleton-less sponges and, more generally, in groups where morphological characters are scarce. Adopting a multi-marker approach is thus highly suitable for these groups.

Molecular phylogeny restores the supra-generic subdivision of homoscleromorph sponges (Porifera, Homoscleromorpha).

BACKGROUND: Homoscleromorpha is the fourth major sponge lineage, recently recognized to be distinct from the Demospongiae. It contains <100 described species of exclusively marine sponges that have been traditionally subdivided into 7 genera based on morphological characters. Because some of the morphological features of the homoscleromorphs are shared with eumetazoans and are absent in other sponges, the phylogenetic position of the group has been investigated in several recent studies. However, the phylogenetic relationships within the group remain unexplored by modern methods. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the first molecular phylogeny of Homoscleromorpha based on nuclear (18S and 28S rDNA) and complete mitochondrial DNA sequence data that focuses on inter-generic relationships. Our results revealed two robust clades within this group, one containing the spiculate species (genera Plakina, Plakortis, Plakinastrella and Corticium) and the other containing aspiculate species (genera Oscarella and Pseudocorticium), thus rejecting a close relationship between Pseudocorticium and Corticium. Among the spiculate species, we found affinities between the Plakortis and Plakinastrella genera, and between the Plakina and Corticium. The validity of these clades is furthermore supported by specific morphological characters, notably the type of spicules. Furthermore, the monophyly of the Corticium genus is supported while the monophyly of Plakina is not. CONCLUSIONS/SIGNIFICANCE: As the result of our study we propose to restore the pre-1995 subdivision of Homoscleromorpha into two families: Plakinidae Schulze, 1880 for spiculate species and Oscarellidae Lendenfeld, 1887 for aspiculate species that had been rejected after the description of the genus Pseudocorticium. We also note that the two families of homoscleromorphs exhibit evolutionary stable, but have drastically distinct mitochondrial genome organizations that differ in gene content and gene order.

An efficient method to find potentially universal population genetic markers, applied to metazoans.

BACKGROUND: Despite the impressive growth of sequence databases, the limited availability of nuclear markers that are sufficiently polymorphic for population genetics and phylogeography and applicable across various phyla restricts many potential studies, particularly in non-model organisms. Numerous introns have invariant positions among kingdoms, providing a potential source for such markers. Unfortunately, most of the few known EPIC (Exon Primed Intron Crossing) loci are restricted to vertebrates or belong to multigenic families. RESULTS: In order to develop markers with broad applicability, we designed a bioinformatic approach aimed at avoiding multigenic families while identifying intron positions conserved across metazoan phyla. We developed a program facilitating the identification of EPIC loci which allowed slight variation in intron position. From the Homolens databases we selected 29 gene families which contained 52 promising introns for which we designed 93 primer pairs. PCR tests were performed on several ascidians, echinoderms, bivalves and cnidarians. On average, 24 different introns per genus were amplified in bilaterians. Remarkably, five of the introns successfully amplified in all of the metazoan genera tested (a dozen genera, including cnidarians). The influence of several factors on amplification success was investigated. Success rate was not related to the phylogenetic relatedness of a taxon to the groups that most influenced primer design, showing that these EPIC markers are extremely conserved in animals. CONCLUSIONS: Our new method now makes it possible to (i) rapidly isolate a set of EPIC markers for any phylum, even outside the animal kingdom, and thus, (ii) compare genetic diversity at potentially homologous polymorphic loci between divergent taxa.

Does hybridization increase evolutionary rate? Data from the 28S-rDNA D8 domain in echinoderms.

The divergent domain D8 of the large ribosomal RNA is very variable and extended in vertebrates compared to other eukaryotes. We provide data from 31 species of echinoderms and present the first comparative analysis of the D8 in nonvertebrate deuterostomes. In addition, we obtained 16S mitochondrial DNA sequences for the sea urchin taxa and analyzed single-strand conformation polymorphism (SSCP) of D8 in several populations within the species complex Echinocardium cordatum. A common secondary structure supported by compensatory substitutions and indels is inferred for echinoderms. Variation mostly arises at the tip of the longest stem (D8a), and the most variable taxa also display the longest and most stable D8. The most stable variants are the only ones displaying bulges in the terminal part of the stem, suggesting that selection, rather than maximizing stability of the D8 secondary structure, maintains it in a given range. Striking variation in D8 evolutionary rates was evidenced among sea urchins, by comparison with both 16S mitochondrial DNA and paleontological data. In Echinocardium cordatum and Strongylocentrotus pallidus and S. droebachiensis, belonging to very distant genera, the increase in D8 evolutionary rate is extreme. Their highly stable D8 secondary structures rule out the possibility of pseudogenes. These taxa are the only ones in which interspecific hybridization was reported. We discuss how evolutionary rates may be affected in nuclear relative to mitochondrial genes after hybridization, by selective or mutational processes such as gene silencing and concerted evolution.