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Most benthic marine invertebrates have complex life cycles that include motile larvae and more or less immobile adult stages. The colonization of surfaces by marine invertebrate larvae is a fundamental process that governs distribution patterns and biodiversity of marine invertebrates. The transition between these life stages is linked to the choice for appropriate habitats via cues for larval settlement and metamorphosis. While marine biofilms have long been recognized as a cue source for larval settlement of phylogenetically diverse invertebrates, the list of chemically identified settlement compounds is surprisingly short. In this chapter, we review the current approach to identify larval settlement cues and discuss the difficulties in differentiating between settlement cues associated with animals or plants and those originating from prokaryotic microbes associated with living surfaces. We further argue for the need to integrate larval ecology, environmental microbiology, analytical chemistry, and surface physics to measure the natural distribution and abundance of larval settlement cues and judge their ecological relevance and plausibility.

Larval settlement of the infaunal spionid polychaetes Polydora cornuta and Streblospio benedicti is mediated by sediment-associated microorganisms. To investigate if larval preference for certain sediment is guided by individual sediment-associated bacteria, 13 bacterial isolates (5 phyla) obtained from the natural habitat of adult polychaetes (Wadden Sea, Germany) in 2008 were screened in still-water, no-choice settlement assays. Two isolates (α-Proteobacterium-Strain DF11 and Flavobacterium-Strain 54) significantly triggered larval settlement in comparison with sterile sediment. In still-water, multiple-choice settlement assays comprising natural and sterile sediment and sediment re-inoculated with isolates DF11 and 54, significant preferences for natural sediment and sediment containing bacterial isolates at 108 cells g−1 were observed. Larval settlement was influenced by bacterial abundance in sediment but the correlation was not strictly positive; thus, maximum larval settlement in response to single bacterial species may occur at certain optimum densities. Non-viable or suspended bacteria and water-soluble bacterial products did not induce larval settlement, suggesting that sediment-associated bacterial settlement cues for P. cornuta and S. benedicti were either produced in situ and/or consisted of heat-labile bacterial products.

Considering that a variety of infaunal polychaetes release halogenated metabolites with potent bactericidal effects into the surrounding sediment, we tested the hypothesis that polychaete- derived halometabolites may either pose a direct negative effect on larvae or alter the bacterial com- munity in surface sediments, and thus indirectly influence larval settlement. Commercially available halogenated compounds identical or similar to the dominant halometabolites found in Streblospio benedicti and Capitella sp. I served as experimental proxies in single- and multiple-choice settlement assays with larvae of S. benedicti. In multiple-choice assays, the hypothesis of a direct influence of halogenated proxies on larval settlement was verified with 1-chlorononane at concentrations of 3.5 µg g -1 sediment. A similar effect was not observed with 2,6-dibromophenol. The no-choice assays did not show a direct influence of halometabolites on polychaete larvae. On the other hand, the hypothesis of an indirect influence of halogenated proxies on larval settlement via modification of the sediment microbial assemblage was clearly rejected. However, an analysis of similarity of the PCR-amplified bacterial genes coding for 16S rRNA corresponding to distinct phylotypes revealed a significant change in bacterial community richness between untreated and natural sediment spiked and incubated with halogenated proxies for 7 d. The change was characterized by the absence of up to 13 phylotypes and the appearance of up to 11 previously undetected phylotypes. The shift in bacterial community richness of sediments exposed to the halogenated proxies was not recognized by larvae, indicating that the halogenated proxies elicited no differential response in larvae of S. benedicti.

The majority of marine invertebrates produce dispersive larvae which, in order to complete their life cycles, must attach and metamorphose into benthic forms. This process, collectively referred to as settlement, is often guided by habitat-specific cues. While the sources of such cues are well known, the links between their biological activity, chemical identity, presence and quantification in situ are largely missing. Previous work on coral larval settlement in vitro has shown widespread induction by crustose coralline algae (CCA) and in particular their associated bacteria. However, we found that bacterial biofilms on CCA did not initiate ecologically realistic settlement responses in larvae of 11 hard coral species from Australia, Guam, Singapore and Japan. We instead found that algal chemical cues induce identical behavioral responses of larvae as per live CCA. We identified two classes of CCA cell wall-associated compounds – glycoglycerolipids and polysaccharides – as the main constituents of settlement inducing fractions. These algae-derived fractions induce settlement and metamorphosis at equivalent concentrations as present in CCA, both in small scale laboratory assays and under flow-through conditions, suggesting their ability to act in an ecologically relevant fashion to steer larval settlement of corals. Both compound classes were readily detected in natural samples.

Most marine invertebrates have dispersive larvae and relatively immobile adults. These developmental stages are linked by a settlement event, which is often mediated by specific cues in bacterial biofilms. While larvae distinguish between biofilms from different environments, it remains unknown if they receive information from all, only a few or even just a single bacterial species in natural biofilms. Here we asked how specific is larval settlement to the bacterial community structure and/or taxonomically distinguishable groups of bacteria in epiphytic marine biofilms? We used novel multivariate statistical approaches to investigate if larval settlement of two sea urchins correlated with the microbial community composition. Larval settlement of Heliocidaris erythrogramma revealed a strong correlation with the community composition, highlighted by canonical analysis of principle components, a constrained ordination technique. Using this technique, the importance of operational taxonomic units (OTUs) within communities relative to larval settlement was investigated. Larval settlement not only correlated, both positively and negatively, with the epiphytic bacterial community composition but also with the relative abundance of few OTUs within these communities. In contrast, no such correlation was observed for the other urchin, Holopneustes purpurascens, whose larvae likely respond to bacterial biofilms in a more general way and specifically respond to a defined settlement cue of algal origin.

Do patchy distribution patterns of infaunal polychaetes result from active site selection of larvae influenced by sedimentassociated microbial cues? This hypothesis was tested with still-water laboratory settlement assays revealing the acceptance or rejection of polychaete larvae to qualitatively different sediments. Laboratory brood cultures of the spionid polychaetes Polydora cornuta and Streblospio benedicti yielded a sufficient number of larvae with planktotrophic development for bioassays. High settlement rates (75–95%) of test larvae were observed in response to natural sediment. Sterilization of natural sediment significantly decreased settlement of P. cornuta (25–55%) while combustion of sediment significantly decreased the settlement rate in both species (5–50%). Differences in settlement responses to sediments treated by sterilization or combustion most likely resulted from a variety of factors such as modified sediment fabric, grain size distribution and quantity of adsorbed organic matter. To experimentally address the potential role of microorganisms and microbial metabolites as mediators of larval settlement, ashed sediment was inoculated with viable microorganisms obtained from natural sediment. In both polychaete species, this treatment significantly increased larval settlement in comparison to the control of ashed sediment indicating that larval settlement was at least partially mediated by the presence of microorganisms associated with sediment.

Various aspects of marine macroinvertebrate ecology cannot be understood without detailed knowledge of larval settlement processes. An important effect underscored during the settlement process is the disturbance of marine invertebrate larvae by predators. We demonstrated that biotic disturbance, comprising physical elimination and mortality due to predation and the behavioral irritation of larvae by the harpacticoid copepod Tisbe japonica, prevent a significant portion of larvae of the polychaete Hydroides elegans from settlement on otherwise suitable substrata. Experiments were performed both in the laboratory and the field showing reproducible significant differences in larval settlement and mortality rates between gender-specific copepod treatments and the control. The trend of decreased larval settlement in the presence of copepods coincided with increased larval mortality in these treatments. In the corresponding field experiments, larval settlement and mortality were similar to the ones obtained under laboratory conditions.

The energetic cost of metamorphosis in cyprids of the barnacle Balanus amphitrite Darwin was estimated by quantification of lipid, carbohydrate and protein contents. About 38–58% (4–5 mJ individual−1) of cypris energy reserves were used during metamorphosis. Lipids accounted for 55–65%, proteins for 34–44% and carbohydrates for

The combined effects of temperature and salinity on larval development and attachment of Balanus trigonus Darwin (Cirripedia, Balanidae) was examined under controlled laboratory conditions. Whilst larval survivorship was not affected (>70%), the duration of larval development was significantly affected by temperature and salinity. The effect of temperature was comparatively stronger than that of salinity. The majority of nauplius II larvae metamorphosed into cypris stage after 4–5 and 10–11 days at 28 °C (22–34‰) and 18 °C (22–34‰), respectively. Temperature, salinity and the duration of assay had a significant effect on cypris attachment with significant interaction among these main effects. Maximum (>80% in 6 days) and minimum percent attachment (0% in 6 days) on polystyrene surfaces were observed at 24 °C (34‰) and 18 °C (22‰), respectively. At high temperature (28 °C) and low salinity (22–26‰), larvae rapidly (4 days) developed into cyprids, but less than 33% attached. These results suggest that low larval attachment rates may lead to the low recruitment of B. trigonus in Hong Kong waters during summer when the water temperature is high (about 28 °C) and salinity is low (

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. ele- gans larval settlement by marine biofilms has been mainly attributed to bacteria and diatoms. In con- trast 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 bio- assays 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 fluores- cein 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.

Nauplii batch cultures of Balanus amphitrite were reared with four different diatoms (Skeletonema costatum, Thalassiosira pseudonana, Chaetoceros gracilis, silicate-limited C. gracilis) at three different cells concentrations: 110 5 ,5 10 5 , and 110 6 cells ml 1 . The cyprid energy reserves were quantified as the ratio of triacylglycerols (TAG) to DNA. Energy reserves of larvae fed on different diatoms at a concentration of 110 6 cells ml 1 were ranked in the order: silicate-limited C. gracilis>C. gracilis>T. pseudonana>S. costatum. There was a significant linear relationship between the TAG content of the diet and cyprid energy reserves. The effect of cyprid energy reserves on metamorphosis to polystyrene surface in the presence and the absence of conspecific settlement factor (SF) was studied after 12, 24, and 48 h of incubation. A strong positive correlation between energy reserves and percent metamorphosis was observed in the absence of SF (r12 h=0.88, r24 h=0.82, r48 h =0.68, P

In order to understand the interaction between extrinsic and intrinsic factors on the metamorphic success of Balanus amphitrite cyprids, we investigated the effect of cyprids energy reserves and physiological age (physiological condition of cyprids) on the metamorphic success under five different combinations of water temperature and salinity mimicking different seasons in subtropical Hong Kong: 18 jC/30x (winter), 22 jC/34x (spring), 28 jC/30x (summer), 28 jC/20x (rainy season), and 25 jC/32x (autumn). The energy reserves of the cyprids were quantified by the ratio of triacylglycerols (TAG) to deoxyribonucleic acid (DNA). The metamorphic success of cyprids was distinctly influenced by their physiological condition and laboratory-mimicked seasons. There was a significant interaction between these factors. In clear contrast to young cyprids with low-TAG/DNA ratio, there was no difference in the metamorphic success of aged cyprids with high-TAG/DNA ratio among seasons. Our results suggest that the physiological condition of cyprids and seasonal variation in water temperature and salinity may jointly determine the metamorphic success of cyprids, and consequently, modulate the

The bacterial component of marine biofilms plays an important role in the induction of larval settlement in the polychaete Hydroides elegans. In this study, we provide experimental evidence that bacterial metabolites comprise the chemical signal for larval settlement. Bacteria were isolated from biofilms, purified and cultured according to standard procedures. Bacterial metabolites were isolated from spent culture broth by chloroform extraction as well as by closed-loop stripping and adsorption of volatile components on surface-modified silica gel. A pronounced biological activity was exclusively observed when concentrated metabolites were adsorbed on activated charcoal. Larvae did not respond to waterborne metabolites when prevented from contacting the bacterial film surface. These results indicate that an association of the chemical signal with a sorbent-like substratum may be an essential cofactor for the expression of biological activity. The functional role of bacterial exopolymers as an adsorptive matrix for larval settlement signals is discussed.

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 -2 . 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.

Various algal diets of different lipid content and composition were used to rear batches of naupliar larvae of Balanus amphitrite. The cyprids in these larval batches differed in lipid content and were used to investigate the combined effect of cyprid lipid content and cyprid age on attachment and metamorphosis. For this purpose, cyprids were aged for 0,3 and 6 d at 8°C prior to utilization in laboratory attachment assays. The percentage attachment of cyprids with similar lipid content differed significantly among the three age categories. A strong and a weak positive relationship between cyprid lipid content and attachment were monitored in young and old cyprids, respectively. A significant interaction (two‐way ANOVA) between cyprid age and lipid content was observed, indicating that these factors jointly affect larval attachment and metamorphosis in B. amphitrite from the beginning of the cyprid stage.

Larval settlement of the barnacle Balanus amphitrite Darwin (Cirripedia, Balanidae) is influenced by natural biofilms. In previous work by others, discriminatory settlement of aged cyprids has been observed in response to biofilms of different age. This study extends prior work by considering the effect of the age of cyprids on the outcome of settlement assays. Settlement was investigated with 0‐day‐old (newly metamorphosed) and 5‐day‐old cyprids. Biofilms under investigation were developed in the field for periods of 5 d and 1 month, and were subsequently included in laboratory settlement assays with a choice between a filmed and an unfilmed substratum. The bioassay was modified from the conventional horizontal dish design in order to generate a low water surface‐to‐volume ratio, which served to suppress larval entrapment in an organic layer on the water surface. Irrespective of cyprid age, a clear discrimination between a filmed and an unfilmed substrata was observed, and the preference for filmed or un...

Field observations in Hong Kong waters have shown the marine macroalga Ulva reticulata Forsskal (Chlorophyta) to be free of fouling. In this study, the presumptive antifouling mechanism in this alga was investigated and attempts were made to distinguish between possible mechanisms. Waterborne algal compounds were analyzed in larval behavioral bioassays with the marine polychaete Hydroides elegans, a major fouling organism in tropical waters around the world. Larval attachment and metamorphosis in this sessile species is induced by specific bacterial strains in natural bio‐organic films. In bioassays with alga‐conditioned seawater, levels of larval metamorphosis were significantly reduced in comparison to controls. The results demonstrate that the inhibitive effect was caused by direct larval deterrence. Following a bio‐assay‐guided isolation procedure, an inhibitive fraction for larval metamorphosis was purified from U. reticulata‐conditioned seawater. Preliminary chemical analysis of the biologically act...

Larval settlement in the serpulid polychaete Hydroides elegans (Haswell) reportedly occurs in response to both an attractive bacterial film and polar, aliphatic dissolved free amino acids (DFAA). In this study, we tested whether the larval metamorphic response was caused by direct DFAA perception or by induction of DFAA-utilizing bacteria in assay dishes. Still-water laboratory assays were performed with sterile measures in the presence and absence of an antibiotic mixture (2.5 × 10 -4 M streptomycin, 1.0 × 10 -4 M penicillin). In the absence of these antibiotics our results revealed a significant correlation between DFAA decrease rates, bacterial film development and larval metamorphosis within a 24 h assay period. After 12 h, DFAA concentrations decreased below the detection limit of 0.013 μM, the attached bacterial density in assay dishes was approx. 2 × 10 4 cells mm -2 , and larval metamorphosis was insignificantly low (20%). After 24 h, DFAA were no longer detectable, the attached bacterial density was unchanged, and larval metamorphosis was significantly high (50 %). In contrast, naturally biofilmed dishes showed a significant metamorphic response of 50 % after 12 h. In analogous experiments in the presence of antibiotics, the percentage of larval metamorphosis varied between 3 and 34% after 24 h while the number of attached bacteria was low (0.5 to 5 × 10 3 cells mm -2 ). We assume that the metamorphic response in these treatments was caused by inductive, antibiotic-resistant bacteria. Our results suggest that larval metamorphosis was exclusively triggered by an inductive bacterial film rather than by direct larval perception of DFAA. Despite most sterile measures at the set-up of bioassays, bacteria were inevitably inoculated into test dishes as they were found associated with the larva and suspended in the larval culture water. Therefore, our results point to a systematic error in this established assay procedure. If putative signaling compounds serve as a nutrition source for larval settlement inducing marine bacteria, we conclude that an explicit investigation of a chemical metamorphic cue's efficacy is unreliable. The ecological significance of the transformation of DFAA into an attractive bacterial film for larval attachment and metamorphosis in H. elegans is discussed.

The early post-settlement stage is the most sensitive during the life history of reef building corals. However, few studies have examined the factors that influence coral mortality during this period. Here, the impact of fouling on the survival of newly settled coral spat of Acropora millepora was investigated by manipulating the extent of fouling cover on settlement tiles using non-toxic, wax antifouling coatings. Survival of spat on coated tiles was double that on control tiles. Moreover, there was a significant negative correlation between percentage cover of fouling and spat survival across all tiles types, suggesting that fouling in direct proximity to settled corals has detrimental effects on early post-settlement survival. While previous studies have shown that increased fouling negatively affects coral larval settlement and health of juvenile and adult corals, to the best of our knowledge, this is the first study to show a direct relationship between fouling and early post-settlement survival for a broadcast spawning scleractinian coral. The negative effects of fouling on this sensitive life history stage may become more pronounced in the future as coastal eutrophication increases. Our results further suggest that targeted seeding of coral spat on artificial surfaces in combination with fouling control could prove useful to improve the efficiency of sexual reproduction-based coral propagation for reef rehabilitation Published version

Biofilms of the bacterium Pseudoalteromonas induce metamorphosis of acroporid coral larvae. The bacterial metabolite tetrabromopyrrole (TBP), isolated from an extract of Pseudoalteromonas sp. associated with the crustose coralline alga (CCA) Neogoniolithon fosliei, induced coral larval metamorphosis (100%) with little or no attachment (0-2%). To better understand the molecular events and mechanisms underpinning the induction of Acropora millepora larval metamorphosis, including cell proliferation, apoptosis, differentiation, migration, adhesion and biomineralisation, two novel coral gene expression assays were implemented. These involved the use of reverse-transcriptase quantitative PCR (RT-qPCR) and employed 47 genes of interest (GOI), selected based on putative roles in the processes of settlement and metamorphosis. Substantial differences in transcriptomic responses of GOI were detected following incubation of A. millepora larvae with a threshold concentration and 10-fold elevated concentration of TBP-containing extracts of Pseudoalteromonas sp. The notable and relatively abrupt changes of the larval body structure during metamorphosis correlated, at the molecular level, with significant differences (p

In the marine environment, biofilms on submerged surfaces can promote or discourage the settlement of invertebrate larvae and macroalgal spores. The settlement-mediating effects of biofilms are believed to involve a variety of biofilm attributes including surface chemistry, micro-topography, and a wide range of microbial products from small-molecule metabolites to high-molecular weight extracellular polymers. The settled organisms in turn can modify microbial species composition of biofilms and thus change the biofilm properties and dynamics. A better understanding of biofilm dynamics and chemical signals released and/or stored by biofilms will facilitate the development of antifouling and mariculture technologies. This review provides a brief account of 1) existing knowledge of marine biofilms that are relevant to settlement mediation, 2) biotechnological application of biofilms with respect to developing non-toxic antifouling technologies and improving the operation of aquaculture facilities, and 3) challenges and future directions for advancing our understanding of settlement-mediating functions of biofilms and for applying this knowledge to real-life situations.

The bacterial component of marine biofilms plays an important role in the induction of larval settlement in the polychaete Hydroides elegans. In this study, we provide experimental evidence that bacterial metabolites comprise the chemical signal for larval settlement. Bacteria were isolated from biofilms, purified and cultured according to standard procedures. Bacterial metabolites were isolated from spent culture broth by chloroform extraction as well as by closed-loop stripping and adsorption of volatile components on surface-modified silica gel. A pronounced biological activity was exclusively observed when concentrated metabolites were adsorbed on activated charcoal. Larvae did not respond to waterbome metabolites when prevented from contacting the bacterial film surface. These results indicate that an association of the chemical signal with a sorbent-like substratum may be an essential cofactor for the expression of biological activity. The functional role of bacterial exopolymers as an adsorptive matrix for larval settlement signals is discussed.

The induction of larval attachment and metamorphosis of benthic marine invertebrates is widely considered to rely on habitat specific cues. While microbial biofilms on marine hard substrates have received considerable attention as specific signals for a wide and phylogenetically diverse array of marine invertebrates, the presumed chemical settlement signals produced by the bacteria have to date not been characterized. Here we isolated and fully characterized the first chemical signal from bacteria that induced larval metamorphosis of acroporid coral larvae (Acropora millepora). The metamorphic cue was identified as tetrabromopyrrole (TBP) in four bacterial Pseudoalteromonas strains among a culture library of 225 isolates obtained from the crustose coralline algae Neogoniolithon fosliei and Hydrolithon onkodes. Coral planulae transformed into fully developed polyps within 6 h, but only a small proportion of these polyps attached to the substratum. The biofilm cell density of the four bacterial strains had no influence on the ratio of attached vs. non-attached polyps. Larval bioassays with ethanolic extracts of the bacterial isolates, as well as synthetic TBP resulted in consistent responses of coral planulae to various doses of TBP. The lowest bacterial density of one of the Pseudoalteromonas strains which induced metamorphosis was 7,000 cells mm(-2) in laboratory assays, which is on the order of 0.1-1% of the total numbers of bacteria typically found on such surfaces. These results, in which an actual cue from bacteria has been characterized for the first time, contribute significantly towards understanding the complex process of acroporid coral larval settlement mediated through epibiotic microbial biofilms on crustose coralline algae.

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 demon- strated to be potent mediators of larval settlement in H. elegans. The putative diatom-derived settle- ment 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 induc- tive (Achnanthes sp., Nitzschia constricta) and non-inductive (Amphora tenerrima, Nitzschia frustu- lum) 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 proteina- ceous 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.