Cell kinetics of the marine sponge Halisarca caerulea reveal rapid cell turnover and shedding.

This study reveals the peculiar in vivo cell kinetics and cell turnover of the marine sponge Halisarca caerulea under steady-state conditions. The tropical coral reef sponge shows an extremely high proliferation activity, a short cell cycle duration and massive cell shedding. Cell turnover is predominantly confined to a single cell population, i.e. the choanocytes, and in this process apoptosis only plays a minor role. To our knowledge, such fast cell kinetics under steady-state conditions, with high turnover by shedding in the absence of apoptosis, has not been observed previously in any other multicellular organism. The duration of the cell cycle in vivo resembles that of unicellular organisms in culture. Morphological and histochemical studies demonstrate compartmentalization of choanocytes in the sponge tissue, which corresponds well with its remarkable cellular kinetics. Coral reef cavity sponges, like H. caerulea, inhabit low nutrient tropical waters, forcing these organisms to filter large volumes of water and to capture the few nutrients efficiently. Under these oligotrophic conditions, a high cell turnover may be considered as a very useful strategy, preventing permanent damage to the sponge by environmental stress. Halisarca caerulea maintains its body mass and keeps its food uptake system up to date by constantly renewing its filter system. We conclude that studies on cell kinetics and functional morphology provide new and essential information on the growth characteristics and the regulation of sponge growth in vivo as well as in vitro and the role of choanocytes in tissue homeostasis.

Sponge paraphyly and the origin of Metazoa.

In order to allow critical evaluation of the interrelationships between the three sponge classes, and to resolve the question of mono- or paraphyly of sponges (Porifera), we used the polymerase chain reaction (PCR) to amplify almost the entire nucleic acid sequence of the 18S rDNA from several hexactinellid, demosponge and calcareous sponge species. The amplification products were cloned, sequenced and then aligned with previously reported sequences from other sponges and nonsponge metazoans and variously distant outgroups, and trees were constructed using both neighbour-joining and maximum parsimony methods. Our results suggest that sponges are paraphyletic, the Calcarea being more related to monophyletic Eumetazoa than to the siliceous sponges (Demospongiae, Hexactinellida). These results have important implications for our understanding of metazoan origins, because they suggest that the common ancestor of Metazoa was a sponge. They also have consequences for basal metazoan classification, implying that the phylum Porifera should be abandoned. Our results support the upgrading of the calcareous sponge class to the phylum level.

[Massive mortality of marine invertebrates: an unprecedented event in northwestern Mediterranean]. / Mortalité massive d'invertébrés marins: un événement sans précédent en Méditerranée nord-occidentale.

An unprecedented mass mortality event has been observed at the end of the summer 1999 along the coasts of Provence (France) and Ligury (Italy). This event has severely affected a wide array of sessile filter-feeder invertebrates from hard-substratum communities, such as sponges (particularly the keratose sponges Hippospongia and Spongia), cnidarians (particularly the anthozoans Corallium, Paramuricea, Eunicella and Cladocora), bivalves, ascidians and bryozoans. Along the Provence coasts, the outbreak spread from east to west. Exceptionally high and constant temperatures of the whole water column (23-24 degrees C, for over one month, down to 40 m) could have determined an environmental context favourable to the mass mortality event. Like the thermal anomaly, the mortality is limited in depth. However, we cannot ascertain whether temperature had a direct effect on organisms or acted in synergy with a latent and/or waterborne agent (microbiological or chemical). Taking into account the global warming context in the NW-Mediterranean, monitoring programs of physical-chemical parameters and vulnerable populations should rapidly be set up.

Phylogenetic analysis of the Hsp70 sequences reveals the monophyly of Metazoa and specific phylogenetic relationships between animals and fungi.

To understand the early evolution of the Metazoa, it is necessary to determine the correct phylogenetic status of diploblastic animals. Despite cladistic studies of morphological characters and recent molecular phylogenetic studies, it remains uncertain whether diploblasts are monophyletic or paraphyletic, and how the phyla of diploblasts are phylogenetically related. The heat shock protein 70 (Hsp70) sequences, because of their ubiquity and high degree of conservation, could provide a useful model for phylogenetic analysis. We have sequenced almost the entire nucleic acid sequence of cytoplasmic Hsp70 from eight diploblastic species. Our data support the monophyly of diploblastic animals. However, the phylogenetic relationships of the diploblast groups were not significantly resolved. Our phylogenetic trees also support the monophyly of Metazoa with high bootstrap values, indicating that animals form an extremely robust clade.

Biological activities of aqueous extracts from marine sponges and cytotoxic effects of 3-alkylpyridinium polymers from Reniera sarai.

We screened the biological activity of 21 marine sponges collected in the northern Adriatic sea. Hemolytic, hemagglutinating, antimicrobial, cytotoxic, and anti-acetilcholinesterase activities of the extracts were monitored. We found that hemolytic activity was generally weak; only extracts from three sponge species possess considerable activity. Hemagglutinating activity was present in almost half of extracts but with little specificity against human erythrocytes of different blood groups. Detectable antimicrobial activity was present in only two extracts, while most of them possessed cytotoxic activity. Strong anti-cholinesterase activity was present only in one sample. 3-alkypyridinium polymers isolated from Reniera sarai were hemolytic and strongly cytotoxic against different cell lines with slightly expressed specificity against transformed cells.

Polyphyly of "sclerosponges" (Porifera, Demospongiae) supported by 28S ribosomal sequences.

To test the competing hypotheses of polyphyly and monophyly of "sclerosponges," sequences from the 5' end of 28S ribosomal RNA were obtained for Astrosclera willeyana, Acanthochaetetes wellsi, and six other demosponge species. Phylogenetic relationships deduced from parsimony and neighbor-joining analyses suggest that these sclerosponges belong to two different orders of Demospongiae: Astrosclera willeyana, being closely related to the Agelasidae, belongs to the Agelasida, Acanthochaetetes wellsi, being closely related to the Spirastrellidae, belongs to the Hadromerida. These results contradict the hypothesis that sclerosponges are monophyletic and imply that a massive calcareous skeleton has evolved independently in several lineages of sponges.

Type IV collagen in sponges, the missing link in basement membrane ubiquity.

Basement membrane structures, or their main component, type IV collagen, have been detected in all multicellular animal species, except sponges. We cancel this exception by the demonstration of type IV collagenous sequences in a new marine sponge species by cDNA and genomic DNA studies. One of these sequences is long enough to demonstrate the specific characteristics of type IV collagen chains. The 12 cysteines are at conserved positions in the carboxyl-terminal non-helical NCl domain, as are the interruptions in the carboxyl-terminal end of the triple helical domain. The gene organization of the region coding for the NCl domain is similar to that of the human genes COL4A2, COL4A4 and COL4A6. An additional, shorter sequence suggests the presence of a second chain. The expected tissue localization of this collagen has been confirmed using polyclonal antibodies raised against a sponge recombinant protein. These results demonstrate that type IV collagen is representated in all animal phyla. It is actually the only known ubiquitous collagen and it has at least two different alpha chains in all the species where it has been characterized.

An analysis of partial 28S ribosomal RNA sequences suggests early radiations of sponges.

Sequences from the 5' end terminal part of 28S ribosomal RNA were obtained and compared for 22 animals belonging to all diploblastic phyla and for a large number of representatives of triploblastic Metazoa and protists. Phylogenetic analyses undertaken using different methods showed deep radiations of phyla such as Ctenophora, Cnidaria and Placozoa but also for groups of Porifera of low taxonomic rank. Short internodes between these radiations suggested an early rapid diversification of diploblasts. A long internal branch preceding the diversification of all triploblasts analyzed could be explained either by a long period with a single ancestor or by the extinction of the earliest triploblastic radiations. Finally some unexpected relationships were revealed among Porifera.

Immunohistochemical studies on the distribution and the function of the D-galactose-specific lectins in the sponge Axinella polypoides (Schmidt).

The distribution of the two D-galactose-specific lectins within the sponge tissue of Axinella polypoides was studied by autoradiography and by an immunohistochemical method on paraplast- and cryosections. Both techniques revealed that the lectins are stored inside the vesicles of the spherulous cells. All spherulous cells, regardless of their appearance in the different types of tissue contained the lectins. Antibodies were purified from an antiserum that reacted with both lectin I and lectin II and from the same antiserum rendered monospecific for lectin I. The purified antibodies were used to demonstrate that lectin II is predominantly present in spherulous cells with small vesicles, and lectin I in those with large vesicles. Electron-microscopic studies revealed that the spherulous cells with small vesicles are derived from archaeocytes and transformed into spherulous cells with large vesicles, a process accompanied by the conversion of lectin II to lectin I. Histological investigations showed that the tips of the bush-like, branched sponge lack the central axis, a spongin fiber network that provides support and stability to the sponge tissue. However, the missing spongin network is already preformed by cell bundles that ultimately produce the numerous fiber strands of the central axis. These bundles are composed exclusively of spindle-shaped cells and the spherulous cells. Other areas where production of spongin fibers is expected are also enriched with spherulous cells. These findings and the reaction of lectin-specific antibodies with the spongin fibers indicate that spherulous cells, and thus the lectins, are involved in synthesis of spongin fiber. Sponges lacking spongin fibers, e.g. Aaptos aaptos and Geodia cydonium, produce lectins with different carbohydrate specificity and possess large numbers of spherulous cells.