E. Richelle-Maurer

Localization and ecological significance of oroidin and sceptrin in the Caribbean sponge Agelas conifera

The Caribbean sponge Agelas conifera was found to produce a mixture of previously described bromopyrrole alkaloids of which oroidin (1) and sceptrin (2) were predominant. This sponge harboured large populations of heterotrophic bacteria but no photosynthetic symbionts (cyanobacteria). However, 1 and 2 were not associated with the bacteria but with the sponge cells as shown by their distribution in enriched cell fractions obtained by differential centrifugation and Ficoll density gradients. Spherulous cells, found in great abundance in the sponge ectosome, were assumed to be involved in the production of 1 and 2. The target compounds were detected, although in small amounts, in short-term cultures of sponge cells, validating the possibility of a continuous cell culture source. Laboratory assays showed that organic sponge extracts affected the behaviour of the coral Madracis mirabilis in causing closure and retraction of the polyps at concentrations of the combined compounds 1 and 2 (1:3.3) as low as 0.7 mg/l (0.0125% of the concentration in whole sponges). At higher concentrations (1.4 mg/l) no recovery of the polyps occurred. The extracts, at almost natural concentrations of 1 and 2, deterred feeding by the predatory reef fish Stegastis partitus, supporting other reported research. In field experiments, wounding induced a sharp increase of 1 and 2 in the sponge tissues but prolonged predator exclusion by caging and forced confrontation with coral neighbours did not yield measurable changes in 1 and 2 concentrations. All sponges were found to release measurable amounts of bromopyrrole alkaloids in seawater conditioned for 30 min. Crude and fractionated sponge extracts and pure sceptrin (2) were active against bacteria, yeast and filamentous fungi. Taken together, these results support a role of oroidin (1) and sceptrin (2) in defence mechanisms against predators and possibly against space competitors and invading and fouling organisms.

Primary cultures from the marine sponge Xestospongia muta (Petrosiidae, Haplosclerida)

In the context of the investigations on the origin and in vitro production of bioactive compounds, primary cultures were developed from ectosomal and choanosomal cell suspensions from the sponge Xestospongia muta. Dissociated cells aggregated and reorganized into a striking reticulated network of cells, typical for X. muta. Moreover, in some cultures an isotropic reticulation of small spicules, very similar to that found in the ectosome of adult sponges, was observed. Phytohaemagglutinin promoted aggregation and the reorganization of the cells. HPLC analyses revealed that straight-chain acetylenic compounds were recovered from short-term cultures and that they were synthesized during culture. Heterotrophic bacteria were assumed to be involved in the process. Together our results established that X. muta would be an excellent experimental model to study, in laboratory conditions, the differentiation of the skeleton and the in vitro biosynthesis of straight-chain acetylenic compounds.

Polyphyly of the genus Axinella and of the family Axinellidae (Porifera: Demospongiaep).

The genus Axinella is difficult to define on the basis of morphological characters and includes a heterogeneous assemblage of species. Several previous authors have suspected the polyphyly of both this genus and the family Axinellidae. To clarify the phylogeny of Axinellidae and Axinella, we propose a new hypothesis based on two molecular markers. In our analyses, Axinellidae and Axinella are polyphyletic assemblages. The 15 species of Axinellidae in our dataset belong to five clades and the nine species of Axinella to three clades. One Axinella clade, named Axinella(p), contains the type-species of the genus: A. polypoides (plus A. aruensis, A. dissimilis, A. infundibuliformis and A. vaceleti). A new clade, Cymbaxinella(p), is proposed, following the PhyloCode, it includes C. damicornis, C. verrucosa, C. corrugata and C. cantharella. The species Axinella cannabina is reallocated to a clade named Acanthella(p). The clades Agelas(p) and Cymbaxinella(p) constitute a new clade: Agelasida(p). Few morphological, biochemical and secondary structures characters support these groupings, highlighting the need for new characters for such problematic sponge groups. This work is an attempt to build a framework for the phylogeny of taxa allocated to Axinella and Axinellidae in the traditional classification.

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 sponges 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.

Homeobox-Containing Genes in Freshwater Sponges: Characterization, Expression, and Phylogeny

The mono- or polyphyletic origin of metazoans has been discussed for a long time because morphological and embryological data are insufficient to discriminate between these alternative hypotheses. The advent of molecular biology and molecular phylogeny with the development of sequencing and the polymerase chain reaction (PCR) has brought some light to this controversy, although molecular data have sometimes led to conflicting interpretations (Field et al. 1988; Lake 1990; Christen et al. 1991; Kobayashi et al. 1993; Wainright et al. 1993; Rodrigo et al. 1994). At present, the monophyly of metazoans is widely accepted, but the phylogenetic position of sponges, considered to be the most primitive metazoan phylum, remains controversial (Muller et al. 1994a; Muller 1995).

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

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 microM, RA had no noticeable influence on sponge development, slightly up-regulating EmH-3 expression. In contrast, in sponges reared in 10, 8 microM and to a lesser extent 2 microM 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.

Conservation and phylogeny of a novel family of non-Hox genes of the Antp class in demospongiae (Porifera)

A survey across the most basal animal phylum, the Porifera, for the presence of homeobox-containing genes led to the isolation of 24 partial or complete homeobox sequences from 21 sponge species distributed in 15 families and 6 orders of Demospongiae. All the new sequences shared a high identity/similarity with EmH-3 (Ephydatia muelleri), a non-Hox gene from the Antp class. The Demox sequences, EmH-3, and related homeodomains formed a well-supported clade with no true affinity with any known bilaterian family, including the Tlx/Hox11 family, suggesting that the EmH-3 family of genes, comprising 31 members, represents a novel family of non-Hox genes, called the Demox family, widespread among Demospongiae. The presence of the Tlx/Hox11 specific signature in the Demox family and common regulatory elements suggested that the Demox and Tlx/Hox11 families are closely related. In the phylogenetic analyses, freshwater Haplosclerida appeared as monophyletic, and Haplosclerida and Halichondrida as polyphyletic, with a clade comprising Agelas species and Axinella corrugata. As for their expression, high levels of Demox transcripts were found in adult tissues. Our data add to the number of published poriferan homeobox sequences and provide independent confirmation of the current Demospongiae phylogenies.