Distribution of brominated compounds within the sponge Aplysina aerophoba:coupling of X-ray microanalysis with cryofixation techniques

The major secondary metabolites of the sponge Aplysina aerophoba are bromin ated compounds. X-ray energy dispersive microanalysis was therefore used to locate secondary metabolites via the Br signal in energy emission spectra from sponge sections. To test the reliability of this method in the face of the loss or redistribution of metabolites during processing, we compared t he results obtained by conventional aldehyde fixation with those obtained b y cryofixation and cryosubstitution with and without cryoembedding. Bromine appeared to be concentrated in two sponge structures, viz, fibres and sphe rulous cells, when cryofixed material was examined. However, X-ray microana lysis failed to demonstrate the presence of bromine in spherulous cells in chemically fixed samples, showing the need for cryotechniques to avoid the loss of compounds. Cryofixation plus cryosubstitution methods performed bes t regarding structural preservation and the immobilization of metabolites. The presence of bromine in the spherulous cells suggests that this cell typ e is the producer of the secondary metabolites, as described for other spon ge species. Nevertheless, the presence of bromine in sponge fibres indicate s that they can accumulate metabolic substances, although we have been unab le to assess whether the chemicals are in their original form or in a modif ied state within the fibres. A. aerophoba has both bacterial and cyanobacte rial symbionts in its mesohyl; the absence of brominated compounds in them contrasts with previous findings in other sponges with prokaryote symbionts .