Until now the bystander effect had only been described in vertebrates. In the present study the existence of this effect has been demonstrated for the phylogenetically oldest metazoan phylum, the Porifera. We used the demosponge Suberites domuncula for the experiments in the two-chamber-system. The lower dish contained irradiated “donor” cells (single cells) and the upper dish the primmorphs (“recipient” primmorphs). The “donor” cells were treated with UV-B light (40 mJ/cm2) and 100 μM hydrogen peroxide (H2O2), factors that exist also in the natural marine aquatic environment of sponges; these factors caused a high level of DNA strand breaks followed by a reduced viability of the cells. If these cells were added to the “recipient” primmorphs these 3D-cell cultures started to undergo apoptosis. This effect could be abolished by the NO-specific scavenger PTIO and ethylene. The conclusion that NO is synthesized by the UV-B/H2O2-treated cells was supported analytically. The cDNA encoding the enzyme dimethylarginine dimethylaminohydrolase (DDAH) was isolated from the “donor” cells. High levels of DDAH transcripts were measured in UV-B/H2O2-treated “donor” cells while after ethylene treatment the steady-state level of expression drops drastically. We conclude that in the absence of ethylene the concentration of the physiological inhibitor for the NO synthase ADMA is low, due to the high level of DDAH. In consequence, high amounts of NO are released from “donor” cells which cause apoptosis in “recipient” primmorphs. In contrast, ethylene reduces the DDAH expression with the consequence of higher levels of ADMA which prevent the formation of larger amounts of NO. This study describes the radiation-induced bystander effect also for the most basal metazoans and demonstrates that this effect is controlled by the two gasses NO and ethylene.

Like in all other Metazoa, also in sponges (Porifera) proliferation, differentiation, and death of cells are controlled by apoptotic processes, thus allowing the establishment of a Bauplan (body plan). The demosponge Lubomirskia baicalensis from the Lake Baikal is especially suitable to assess the role of the apoptotic molecules, since its grade of construction is highly elaborated into an encrusting base and branches composed of modules lined up along the apical-basal axis. The four cDNAs, ALG-2, BAK, MA-3, and Bcl-2, were isolated from this sponge species. The expression levels of these genes follow characteristic gradients. While the proapoptotic genes are highly expressed at the base of the branches and comparably low at the top, the pro-survival gene follows an opposite gradient. Parallel with the tuned expression of these genes, the activities of the apoptosis-executing enzymes caspase-8 (IETDase activity) and caspase-3 (DEVDase activity) are lowest at the top of the branch and highest at their base. This characteristic expression/activity pattern of the genes/enzymes, which had been determined in a few specimens, collected from an unpolluted, natural site, appears reversed in specimens collected from an anthropogenically polluted site. These findings indicate the involvement of apoptotic proteins in the axis formation (branches) in L. baicalensis.

Selenium is a trace element found in freshwater and the marine environment. We show that it plays a major role in spicule formation in the demosponge Suberites domuncula. If added to primmorphs, an in vitro sponge cell culture system, it stimulates the formation of siliceous spicules. Using differential display of transcripts, we demonstrate that, after a 72‐h exposure of primmorphs to selenium, two genes are up‐regulated; one codes for selenoprotein M and the other for a novel spicule‐associated protein. The deduced protein sequence of selenoprotein M (14 kDa) shows characteristic features of metazoan selenoproteins. The spicule‐associated protein (26 kDa) comprises six characteristic repeats of 20 amino acids, composed of 10 distinct hydrophobic regions (≈ 9 amino acids in length). Recombinant proteins were prepared, and antibodies were raised against these two proteins. Both were found to stain the central axial filament, which comprises the silicatein, as well as the surface of the spicules. In the presence of selenium, only the genes for selenoprotein M and spicule‐associated protein are up‐regulated, whereas the expression of the silicatein gene remains unchanged. Finally we show that, in the presence of selenium, larger silica aggregates are formed. We conclude that selenium has a stimulatory effect on the formation of siliceous spicules in sponges, and it may be involved in the enzymatic synthesis of biosilica components.

Silicatein immobilised on self-assembled polymer layers using a histidine-tag chelating anchor group retains its hydrolytical activity for the formation of biosilica, and catalyses the formation of layered arrangements of biotitania and biozirconia.