Larval settlement in the fouling polychaete Hydroides elegans Haswell (1883) is mediated by natural biofilms, a complex consortium of marine bacteria, benthic diatoms, protozoa, fungi and adsorbed organic matter. Currently, the inductive effect of natural biofilms on larval settlement is attributed to the bacterial component. In this paper, we present results of an investigation of the diatom component of natural biofilms with respect to induction of larval settlement in H. elegans. Thirty-two diatom strains were isolated from natural biofilms, identified and individually investigated in still-water laboratory settlement assays. A multiple pairwise comparison of larval settlement rates grouped the diatoms into 3 statistical categories: (1) 'inductive'; same percentage of larval settlement as in the positive control (glass slide with natural biofilm); (2) 'no effect'; same percentage of larval settlement as in the negative control (clean glass slide); and (3) 'weakly inductive'; percentage of larval settlement significantly higher and lower than in the negative and the positive control, respectively. Out of 32 diatom strains, 3 (10%) fell into the first, 19 (59%) into the second and 10 (31%) into the third category. The variability in larval settlement was not correlated with the density of diatoms in the monospecific films. Despite sterile measures and the use of antibiotics, diatom films under investigation were not axenic, with bacterial abundances ranging from 200 to 8000 cells mm Ϫ2. No quantitative correlation between the bacterial abundance in diatom films and the percentage of larval settlement was observed. To evaluate the qualitative impact of the bacterial fraction in the mixed diatom-bacteria films on the induction of larval settlement, 6 representative diatom strains, comprising the 3 categories stated above, were selected to create diatom-free microbial films with the bacterial contaminants of each culture. Whilst the bacterial abundance in the non-axenic diatom films and the diatom-free control films was statistically the same, the percentage of larval settlement towards these treatments differed significantly for the diatom strains belonging to category (1) and (3). These categories comprised 4 different genera of diatoms, indicating that the observed inductive effect on larval settlement was not restricted to a certain diatom genus. Our results suggest that in addition to microbial films, benthic diatoms may significantly contribute to the mediation of larval settlement behaviour in H. elegans by marine biofilms.

Larval settlement of the polychaete Hydroides elegans Haswell, 1883 is mediated by marine biofilms; complex agglomerates of bacteria, diatoms, fungi and protozoa enmeshed in a matrix of extracellular polymers (EPS). In our previous investigations, benthic diatoms were demonstrated to be potent mediators of larval settlement in H. elegans. The putative diatom-derived settlement cues were heat-stable components in close association with the diatom surface. For an in-depth investigation of the chemical nature of diatom-derived larval settlement cues, the EPS of the inductive (Achnanthes sp., Nitzschia constricta) and non-inductive (Amphora tenerrima, Nitzschia frustulum) diatoms were bioassayed for their effect on larval settlement. When EPS fractions larger than 100 kDa were immobilized in stable hydrogels to mimic their association to a solid substratum, they evoked an effect on larval settlement similar to that of the respective monospecies diatom films. The crude exopolymer samples mainly consisted of polysaccharides with a small proportion of proteinaceous sample components. After enzymatic removal of proteinaceous EPS components, the samples evoked an effect on larval settlement similar to that of the crude EPS samples, indicating the negligible role of large proteins or glycoproteins as settlement cues. KEY WORDS: Diatoms · Larvae · Settlement · Biofilm · Exopolymers · Hydroides elegansResale or republication not permitted without written consent of the publisher

Larval settlement in the polychaete Hydroides elegans is mediated by marine biofilms, which are complex agglomerates of bacteria, diatoms, fungi and protozoa. The induction of H. elegans larval settlement by marine biofilms has been mainly attributed to bacteria and diatoms. In contrast to bacteria-derived settlement cues, the nature and origin of diatom-derived settlement cues is poorly understood. In this study, we present the first investigation on the nature and origin of larval settlement cues produced by marine diatoms. Diatoms with inductive (i.e. Achnanthes sp. and Nitzschia constricta) and non-inductive (i.e. Amphora tenerrima and N. frustulum) effects on larval settlement of H. elegans were selected as model strains in this investigation. Larval settlement bioassays with a choice between monospecies diatom films and unfilmed substratum revealed that the diatom-derived settlement cue was water-insoluble and associated with the film surface. There was a clear correlation between the surface coverage of diatoms in films and their inductive effect on larval settlement. In the case of Achnanthes sp., even the lowest surface coverage of 1.8% induced larval settlement significantly more than the control of filtered seawater. The interstitial distance between diatom cells at this low film density was within the body size range of H. elegans larvae (100 ± 30 µm). The inductive effect of diatom films on larval settlement prevailed even after heat treatments (121°C for 1 h) that completely killed the diatoms, as verified with the vital stain fluorescein diacetate. These results suggest that the induction of larval settlement by diatoms is not related to their viability, and that, contrary to marine bacteria, diatom-induced larval settlement cues are composed of heat-stable surface components, such as capsular extracellular polysaccharides. These results not only demonstrate that different components of biofilms play inductive and inhibitive roles on larval settlement, but also that their relative space occupation deserves consideration regarding their roles as mediators of larval settlement. KEY WORDS: Diatom · Larvae · Settlement · Biofilm · Hydroides elegansResale or republication not permitted without written consent of the publisher

Many macroalgae employ chemical means to suppress epibiosis by micro-and macroorganisms. Previous studies have focused on the effects of tissue extracts of entire algae on a few bacterial isolates, thereby missing not only ecologically relevant bacteria but also natural delivery mechanisms of algal antimicrobial agents. In this study, we investigated the potential antimicrobial effects of waterborne macroalgal metabolites utilizing a cultureindependent approach to compare the bacterial community richness in seawater in the presence and absence of macroalgae. The methodology comprised the collection of planktonic bacteria in algal culture water on membrane filters followed by filter PCR and denaturant gradient gel electrophoresis (DGGE) of 16S rRNA gene sequences of harvested bacteria with universal primers. Similarity analysis distinguished two groups of macroalgae under investigation, one of which showed >55% difference, and the other <50% difference in bacterial community composition in comparison to natural seawater. The bacterial abundance in algal culture water of different algae was reduced between 20% and 50%. Further experiments demonstrated that the observed effects were caused by waterborne algal compounds. However, some bacterial types were exclusively eliminated in the presence of algae, indicating causative modes of action other than direct exposure of bacteria to waterborne compounds, such as surfacemediated antimicrobial effects.

To test whether macroalgae affect microbial colonizers in close proximity in a phylum-specific fashion, the community richness of planktonic bacteria and fungi was analyzed with selective oligonucleotide probes targeting the Cytophaga/Flavobacterium/Bacteroides (CFB), Alphaproteobacteria and Roseobacter group and the ITS1 region of marine fungi. Naturally occuring planktonic microorganisms were incubated in the presence of macroalgae or in seawater previously conditioned with macroalgal metabolites. The red algae Ceramium rubrum and Mastocarpus stellatus as well as seawater conditioned with these algae reduced the community composition of bacteria to a greater extent than the brown alga Laminaria digitata, indicating that metabolites differed among macroalgae or that the susceptibility of planktonic bacteria towards alga-derived antimicrobials correlated with their phylogenetic affiliation. The most affected phylotypes belonged to the CFB and the Roseobacter clade. The planktonic fungal community was only affected in the presence of macroalgae and not in algal-conditioned water, but with a specificity different from that observed for bacteria. The macroalgae L. digitata and M. stellatus exhibited more pronounced antifungal effects than C. rubrum. This study demonstrates macroalgal defenses against epiphytic microorganisms based on natural delivery mechanisms of allelochemicals utilizing a culture-independent approach, thus minimizing the ecological bias inherent to culture-dependent studies based on few microbial isolates.