Earlier studies have shown that biofilms can mediate the larval settlement of the polychaete Hydroides elegans and that changes in the bacterial community structure and density of biofilms often alter the larval settlement response. However, the chemical cues that mediate this response remain unknown. In this study, both successional changes in the bacterial community structure and the chemical profiles of subtidal biofilms are described and related to the larval settlement response. Multispecies biofilms were developed on polystyrene Petri dishes and granite rock in the subtidal zone over a period of 20 days. The effects of the substratum and age on the bacterial community structure and chemical profiles of the biofilms were evaluated with two molecular methods (microarray (PhyloChip) and denaturing gradient gel electrophoresis) and with gas chromatography-mass spectrometry, respectively. Both age and substratum altered the bacterial community structures and chemical profiles of the biofilms. Age had a greater effect in shaping the bacterial community structure than did the substratum. In contrast, the type of substratum more strongly affected the chemical profile. Extracts of biofilms of different ages, which developed on different substrata, were tested for the settlement of H. elegans larvae. The extracts induced larval settlement in a biofilm-age-dependent manner, and extracts originating from different substrata of the same age showed no differences in larval settlement. Our results suggest that the larval settlement response cannot be predicted by the overall chemical composition of the biofilm alone.

We studied the effect of the quorum-sensing (QS) blockers 5-hydroxy-3[(1R)-1-hydroxypropyl]-4-methylfuran-2(5H)-one (FUR1), (5R)-3,4-dihydroxy-5-[(1S)-1,2-dihydroxyethyl]furan-2(5H)-one (FUR2) and triclosan (TRI) on the formation of bacterial biofilms, and the effect of these biofilms on the larval attachment of the polychaete Hydroides elegans and the bryozoan Bugula neritina. 14-day-old subtidal biofilms were harvested from artificial substrata and were allowed to develop in the laboratory with and without QS blockers. QS blockers inhibited the production of violacein by the QS reporter strain Chromobacterium violaceum CV026 and did not affect the metabolic activity of bacteria in multispecies biofilms. At a concentration of 10(-3) M all three tested compounds inhibited the establishment of microbial communities, but at one of 10(-4) M only FUR2 inhibited establishment. The tested QS blockers caused changes in bacterial density and bacterial community structure, as revealed by terminal restriction fragment length polymorphism and FISH. The groups most affected by QS blockers were Alphaproteobacteria, Gammaproteobacteria and the Cytophagales. Larvae of H. elegans and B. neritina avoided settling on biofilms that had developed in the presence of QS blockers. Our results suggest that QS blockers directly control the formation of multi-species biofilms, and indirectly - by means of biofilm properties - affect larval attachment on these modified biofilms.

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