Effects of environmental factors on biofilms and subsequent larval attachment of benthic marine invertebrates

Many studies have shown that biofilms on marine surfaces are a major source of attachment cues for larvae of a broad range of marine invertebrates. The abundance and composition of microorganisms in biofilms are critically controlled by the surrounding environments. Any factors that can alter biofilms may indirectly affect larval attachment and thus recruitment. However, study on the interactions among environmental heterogeneity, biofilms and larval attachment is limited. The main goal of this thesis research was to investigate the effects of environmental factors on biofilms and subsequent larval attachment of benthic marine invertebrates. The focuses of this study were on: 1) the interactive effects between surface wettability and biofilms on the barnacle Balanus amphitrite larval attachment in order to investigate if the substrata used for the biofilm studies would alter the positive effect of natural biofilms on larval attachment; 2) the effects of ultraviolet radiation on biofilms and subsequent larval attachment of the intertidal species barnacle B. amphitrite and the subtidal species polychaete Hydroides elegans; 3) the attachment response of B. amphitrite larvae to spatial-temporal variations in biofilms; and 4) the characterization of inductive chemical cues from natural biofilms (i.e. biofilms selected from previous study that induced the highest larval attachment). Surface wettability was demonstrated to affect the attractiveness of the substratum to barnacle Balanus amphitrite larval attachment. Larvae preferred to attach to a high wettability than to a low wettability surface regardless of the cyprids age. On the other hand, biofilm ages (i.e. different bacterial community compositions) were shown to affect larval attachment. For example, larvae preferred mature (i.e. 6-d-old) biofilms over young biofilms i.e. 3-d-old) for attachment, irrespective of the type of substrata. This result indicated that surface wettability does not alter the positive effect of natural biofilms on larval attachment. Ultraviolet radiation, both UV-A and UV-B, caused a decrease in the percentage of metabolically active bacterial cells in biofilms. UV-B caused a greater loss of metabolically active bacterial cells than UV-A at the same energy level. The reduction in densities of metabolically active bacteria did not affect the attachment preference of the barnacle Balanus amphitrite larvae, while larval attachment of the polychaete Hydroides elegans in response to biofilms decreased in a UV-dose-dependent manner. This result indicated that bacterial metabolic activity is required for the biofilms to have an inductive or inhibitive effect for larval attachment of the subtidal species H. elegans, but not for the intertidal species B. amphitrite. This two opposing results indicated that biofilms and larval interactions are complex and highly species specific, as larvae of different species respond differently to chemical cues derived from biofilms. Using culture independent techniques, I demonstrated that the bacterial community composition of biofilms originating from different sites were different. In addition, larvae of Balanus amphitrite preferred to attach to biofilms originating from habitats where recruitment, juvenile growth and survival were the highest. This result indicated that spatial variation in the bacterial community composition of biofilms might lead to differential attachment of barnacle larvae, and thus recruitment. Natural biofilm crude extract was demonstrated to induce the larval attachment of Hydroides elegans. Using bioassay guided fractionation, the non-polar fraction of natural biofilm crude extract was shown to induce a higher percentage of larval attachment than the polar fraction. In addition, two inductive compounds were obtained by further purification using high performance liquid chromatography (HPLC) and chemical characterization using nuclear magnetic resonance (NMR). One of the active compounds was identified as octadecenoic acid with 18 carbons