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Biofilm microbial communities play an important role in the larval settlement response of marine invertebrates. However, the underlying mechanism has yet to be resolved, mainly because of the uncertainties in characterizing members in the communities using traditional 16S rRNA gene-based molecular methods and in identifying the chemical signals involved. In this study, pyrosequencing was used to characterize the bacterial communities in intertidal and subtidal marine biofilms developed during two seasons. We revealed highly diverse biofilm bacterial communities that varied with season and tidal level. Over 3,000 operational taxonomic units with estimates of up to 8,000 species were recovered in a biofilm sample, which is by far the highest number recorded in subtropical marine biofilms. Nineteen phyla were found, of which Cyanobacteria and Proteobacteria were the most dominant one in the intertidal and subtidal biofilms, respectively. Apart from these, Actinobacteria, Bacteroidetes, and Planctomycetes were the major groups recovered in both intertidal and subtidal biofilms, although their relative abundance varied among samples. Full-length 16S rRNA gene clone libraries were constructed for the four biofilm samples and showed similar bacterial compositions at the phylum level to those revealed by pyrosequencing. Laboratory assays confirmed that cyrids of the barnacle Balanus amphitrite preferred to settle on the intertidal rather than subtidal biofilms. This preference was independent of the biofilm bacterial density or biomass but was probably related to the biofilm community structure, particularly, the Proteobacterial and Cyanobacterial groups.

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. The ISME Journal (2010) 4, 817-828; doi:10.1038/ismej.2009.157; published online 21 January 2010

Earlier studies have shown that biofilms can mediate larval settlement of many benthic invertebrates including the polychaete Hydroides elegans and the barnacle Balanus amphitrite; changes in bacterial community structure and other biofilm attributes influence the larval settlement response. However, the chemical cues mediating the settlement response remain unknown. Molecular tools: Phylochip, denaturing gradient gel electrophoresis (DGGE), pyrosequencing and clone library construct, and chemical analytic tools: gas chromatography-mass spectrometry (GC-MS) and ultra performance liquid chromatography-mass spectrometry (UPLC-MS) were used to provide in-depth analysis on bacterial community structure and chemical profile of biofilms at different environmental conditions. Age showed a greater effect on shaping the bacterial community structure but less effect on chemical profile than substratum did. However, biofilm extracts induced larval settlement of H. elegans in a biofilm-age-dependent manner, suggesting that specific settlement cues are involved and the larval settlement response cannot be predicted by the overall chemical composition of the biofilm alone. Pyrosequencing revealed extremely high biodiversity in biofilms ever reported and a high portion of rare population. Bacterial community comparison between intertidal and subtidal biofilims indicated that Cyanobacteria were more abundant in intertidal biofilms whereas Proteobacteria were more abundant in subtidal biofilms. Moreover, I demonstrated that larval settlement of B. amphitrite is a chemically-mediated process by chemical choice assay. Principal components analysis (PCA) and preliminary compound matching indicated that the major difference in chemical profile between intertidal and subtidal biofilms is the presence of some amino acid derivatives and this may explain the preferential settlement of barnacle in the intertidal zone.

Copper (Cu) contamination is a potential threat to the marine environment due to the use of Cu-based antifouling paints. Cu stress on larval settlement of the polychaete Hydroides elegans was investigated, and this was linked to Cu stress on biofilms and on the biofilm development process. The inductiveness of young biofilms was more easily altered by Cu stress than that of old biofilms, indicating the relative vulnerability of young biofilms. This might result from changes in bacterial survival, the bacterial community composition and the chemical profiles of young biofilms. Cu also affected biofilm development and the chemical high performance liquid chromatograph fingerprint profile. The results indicate that Cu affected larval settlement mainly through its effect on the process of biofilm development in the marine environment, and the chemical profile was crucial to biofilm inductiveness. It is strongly recommended that the effects of environmentally toxic substances on biofilms are evaluated in ecotoxicity bioassays using larval settlement of invertebrates as the end point.

Multi-species natural microbial biofilms have been identified as sources of cues to induce larval attachment of a broad range of sessile marine invertebrates. However, the chemical identities of the cues originating from these films have not been fully characterized. In this study, we isolated and characterized 2 compounds from natural biofilms that induced the larval attachment of the polychaete Hydroides elegans. Biofilms were developed on Petri dishes in seawater in the field for 6 d, collected from the Petri dishes using cotton buds, and extracted using a combination of polar and non-polar solvents. The non-polar fraction was inductive to larval attachment and was thus further fractionated using HPLC. Bioassay-guided fractionation was used to locate the active compounds that were then structurally characterized by using nuclear magnetic resonance (NMR) spectroscopy. Two inductive compounds were identified, a hydrocarbon (6,9-heptadecadiene) and a fatty acid (12-octadecenoic acid), which are the first chemical cues inductive to larval attachment of marine invertebrates isolated from natural biofilms.