Induction Of Settlement Research Articles

Effect of Biofilm Formation by Pseudoalteromonas spongiae on Induction of Larval Settlement of the Polychaete Hydroides elegans

The effects of culture conditions and chloramphenicol treatment on the induction of the marine bacterium Pseudoalteromonas spongiae to larval settlement of Hydroides elegans were investigated. The results showed that P. spongiae cells grown in the medium containing both yeast extract and peptone (YP-grown P. spongiae) was highly inductive to larval settlement, whereas P. spongiae cells grown in the medium containing only peptone (P-grown P. spongiae) or YP-grown P. spongiae cells treated with chloramphenicol at the onset of biofilm development (YPC-grown P. spongiae) did not induce larval settlement. Analysis of biofilm formation, biofilm structure, and the surface protein profile indicated that only the induction-capable YP-grown P. spongiae formed a well-developed biofilm, while the P-grown P. spongiae and the YPC-grown P. spongiae did not. We report here for the first time that bacterial biofilm formation was associated with its induction of larval settlement.

FACTORS AFFECTING SETTLEMENT OF ABALONE (HALIOTIS IRIS) LARVAE ON BENTHIC DIATOM FILMS

Diatoms are widely used in abalone hatcheries to induce larval settlement (=attachment and metamorphosis) but there are few data on the factors that influence their effectiveness as settlement cues. Of 16 diatom strains tested in Experiment 1, half induced ≥80% attachment and >50% metamorphosis within 4 days. Settlement success did not correlate significantly with diatom abundance or adhesive strength (P > 0.05). Several diatom strains interfered with settlement. Examples included (1) smothering by highly mobile diatoms (Nitzschia longissima); (2) shells becoming stuck to sticky secretions (Navicula britannica, Achnanthes longipes); and (3) unstable diatom cells preventing pedal attachment (Licmophora sp.). In Experiment 2, only 1 of 15 diatom strains induced over 70% metamorphosis, and three others induced 22–36% metamorphosis, within 4 days. Older cultures of a strain induced higher attachment and metamorphosis than younger cultures of the same strain (P < 0.001), but some old cultures still gave poor settlement. For the young diatom cultures, larval attachment correlated positively with diatom percent cover (r = 0.89, P < 0.05), and metamorphosis with the growth phase of the diatom film (r = 0.91, P < 0.05). Experiments 3–5 examined the role of bacteria in settlement induction by diatom films. In Experiment 3, films of 8 diatom species from Experiment 1 were regrown from ∼6 individually isolated and rinsed cells, likely altering the associated bacterial flora. Metamorphosis was 179 fold lower on average than in Experiment 1 (P < 0.0001), but attachment and diatom density were not significantly different (P = 0.87 and P = 0.75 respectively). In Experiment 4, Nitzschia ovalis grown and assayed with antibiotics had lower metamorphosis after 2 days than the same strain grown and assayed without antibiotics (6 vs 64%, P = 0.004), whereas attachment after 2 days did not differ (94 versus 92%, P = 0.49). In Experiment 5 bacteria from a Nitzschia ovalis culture induced as much attachment and metamorphosis as the intact diatom film, whereas cell-free supernatant was much less effective. The presence of antibiotics in settlement assays reduced attachment and metamorphosis by biofilms, but not by coralline algae (Phymatolithon repandum) or GABA. In Experiment 5 metamorphosis on diatoms and bacteria occurred gradually over 2 wk, whereas on coralline algae and GABA it occurred within a few days. This study suggests that many diatoms cue rapid larval attachment, but few induce consistently strong metamorphosis within 4 days in laboratory conditions. The bacteria present in diatom films affect the settlement-inducing activity of the film in at least some cases. The effectiveness of diatom films was generally higher for more mature films but overall, physical factors explained little of the variation in the activity of diatoms.

Larval settlement and metamorphosis of the mussel Mytilus galloprovincialis on different macroalgae

Settlement of mussels is commonly associated with macroalgae. The effects of 19 macroalgal species on the settlement and metamorphosis of pediveliger larvae of the mussel Mytilus galloprovincialis were investigated in the laboratory. Settlement and metamorphosis inducing activities of macroalgae Chlorodesmis fastigiata and Ceramium tenerrimum collected each month during the period between January 2005 and April 2006 were also investigated. Furthermore, C. fastigiata and C. tenerrimum were subjected to various treatments to investigate the roles of bacteria and diatoms on the algal surface in the induction of larval settlement and metamorphosis of M. galloprovincialis and the characteristics of the cues in these two macroalgae. Pediveliger larvae of M. galloprovincialis settled and metamorphosed in high percentages on Cladophora sp., Chlorodesmis fastigiata, Centroceras clavulatum, and Ceramium tenerrimum, all of which were filamentous in morphology. Macroalgae that were cylindrical, phylloid, flabellate, palmate and pinnate all showed low (<8%) percentages of post-larvae but four other filamentous macroalgae also had low mussel larval settlement, suggesting that chemical factors may also be involved. Seasonal variation had no effect on inductive activities of C. fastigiata and C. tenerrimum. Treatment of C. fastigiata and C. tenerrimum with formalin, ethanol and heat resulted in the significant decrease or loss of their inductive activities. Survival of algal cells within treated macroalgae also decreased significantly. Treatment of the two macroalgae with antibiotics and GeO2 reduced the numbers of bacteria and diatoms on their surface but did not affect their inductive activities, indicating that the cue was produced by macroalgae and not by coexisting bacteria and diatoms. However, conditioned water and crude extracts of these two macroalgae did not induce larval settlement and metamorphosis. Thus, larvae of M. galloprovincialis settled and metamorphosed on specific filamentous macroalgae. The chemical cues produced by C. fastigiata and C. tenerrimum were susceptible to ethanol and heat treatments and were not recovered in the conditioned water nor in the extracts. The finding that inactive C. tenerrimum can be produced from culturing its apical segments provides a new tool to elucidate the chemical cue(s) from macroalgae through manipulation of their culture conditions.

Individual-based model of larval transport to coral reefs in turbulent, wave-driven flow: behavioral responses to dissolved settlement inducer

Larvae of many benthic marine animals set- tle and metamorphose in response to waterborne chem- ical cues. Can the behavioral responses of microscopic larvae in the water column to dissolved chemical cues affect their transport to the substratum in the turbulent, wave-driven flow characteristic of many shallow coastal habitats? We addressed this question using an individ- ual-based model of larvae of the sea slug Phestilla sibo- gae, transported in the oscillatory flow above coral reefs. Larvae of P. sibogae stop swimming and sink in response to a dissolved inducer released by their prey, the coral Porites compressa, and resume swimming when exposed to inducer-free water. The instantaneous fine-scale spa- tial distribution of inducer in the flow above a reef is fila- mentous; hence microscopic larvae swimming or sinking through the water encounter inducer above threshold concentration in on/off temporal patterns. Model results show that using a time-averaged inducer concentration gradient to calculate larval transport rates to the reef overestimates the rates by <15% (depending on the threshold concentration of inducer required to trigger larval sinking) compared with those calculated using time-varying, fine-scale inducer distributions. Aspects of larval behavior that have large effects on rates of trans- port to the substratum are swimming speed and direc- tion, sinking speed, and sensitivity (threshold concentra- tion) and responsivity (percent of encounters eliciting a response) to inducer. In contrast, lag times to start sinking after encountering inducer or to resume swimming after re-entering inducer-free water, have negligible effect.

Settlement of larval top shell Turbo�(Batillus)�cornutus in response to several marine algae

The settlement of larval top shell Turbo (Batillus) cornutus in response to seven macroalgal and three benthic diatom species was examined in the laboratory. The percentages of larval settlement different significantly among algal species, and were significantly higher on two red algal species (Marginisporum crassissima, an articulated coralline alga, and Gelidium elegans, an agarophyte) than on the green algae, brown algae and benthic diatoms tested. Marginisporum crassissima induced larval settlement most strongly throughout the experiments. The percentage of larval settlement on a crustose coralline alga (unidentified species), which is known as a suitable substratum for abalone larvae, was much lower than on M. crassissima and G. elegans, which have morphologically complex growth forms. The percentage of settled individuals on ethanol-killed M. crassissima was much lower than on intact M. crassissima. The results indicate that physical structure is unlikely to be the main inducer of settlement; therefore, settlement of top shell larvae seems to be strongly induced by chemicals from the two red algal species. The specific distribution of juvenile top shell in the field may be at least partly determined by the strong induction of larval settlement by articulated coralline and gelidiacean algae.

Presence of Acyl-Homoserine Lactone in Subtidal Biofilm and the Implication in Larval Behavioral Response in the Polychaete Hydroides elegans

Quorum sensing (QS) signals have been considered to play important roles in biofilm development and in the attractiveness of biofilms to higher organisms in marine ecosystem. In this study, bacterial QS signalsacylated homoserine lactone derivatives (AHLs) were detected in 2-, 4-, and 6-day-old subtidal biofilms by using AHLs reporter strains. N-dodecanoyl-homoserine lactone (C12-HSL) was identified in 6-day-old biofilm at a concentration of 9.04 microg cm(-minus;2) (3.36 mmol l(-minus;1)). To investigate the possible role of AHLs in the consequent eventlarval settlement of the polychaete Hydroides elegans onto subtidal biofilmsseven biofilm-derived bacteria that effectively induced larval settlement of H. elegans, were screened for AHL production. One of them, the Vibrio sp. UST950701-007, produced N-hexanoyl-homoserine lactone (C6-HSL). Larval settlement bioassay showed that C6-HSL, C12-HSL, and 3-oxo-octanoyl-homoserine lactone (3-oxo-C8-HLS) at certain concentrations induced some initial larval settlement behaviors such as reducing swimming speed, crawling on the bottom. However, these AHLs did not effectively induce larval settlement in comparison to the effective settlement inducer 3-isobutyl-1-methylxanthine. The possible chemokinetic mechanism and indirect effects of AHLs on larval settlement are suggested.

Effect of epinephrine on the settlement and metamorphosis of Manila clam larvae

Chemical inducement and DDRT-PCR (differential display reverse transcription PCR) are adopted to investigate the effect of epinephrine (EPI) on the settlement and metamorphosis of Manila clam larvae. Chemical inducement shows that EPI has an effect to some extent on the metamorphosis of Manila clam larvae at all concentrations and in all treatments designed. The most significant result of inducement is obtained at the concentration of 10−6 mol L−1 and for 4h. DDRT-PCR using six primer pairs shows that the gene expression pattern is quite different between EPI treatment and the control. Three hundred and forty-three amplification bands are obtained in total, among which, 67 (19.53%) are differentially appeared. Therefore, EPI has an effect on the gene expression of the eye spot larval Manila clam. It can be hypothesized that EPI is a settlement and metamorphosis inducer for Manila clam. EPI may lead to larvae settlement and metamorphosis by binding to the receptors on the membrane and then changing the gene expression of larvae cells.

Lipid class dynamics during larval ontogeny of sea scallops, Placopecten magellanicus, in relation to metamorphic success and response to antibiotics

This study examines ontogenetic changes in lipid class composition in relation to survival and growth of five batches of sea scallops, Placopecten magellanicus, from egg to dissoconch stages. It also investigates the effects of antibiotic addition during metamorphosis to determine whether transient treatment during this critical period is effective in increasing metamorphic success. The top-performing larvae (growth rate: 4.7-5.0 μm day − 1 , dissoconch survival: 32–57%) were characterized by a pronounced increase in absolute (ng larva − 1 ) and relative concentrations (wt.% of total lipid) of triacylglycerol (TAG) during the pre-metamorphic period, followed by utilization during metamorphosis (0.5–1.4 μg day − 1 larva − 1 ). In contrast, the low-performing scallops (growth rate: 3.6–4.5 μm day − 1 , dissoconch survival < 1%) exhibited a constant, low level of TAG. These results strongly suggest that the accumulation of TAG during the pre-metamorphic period is a good predictor of scallop performance as measured by survival to dissoconch stage. Antibiotic treatment enhanced absolute and relative TAG concentrations in pre-metamorphic scallops and resulted in higher post-metamorphic survival and TAG concentration than in non-treated controls. However, the proportion of dissoconch to live larvae at the end of the experiment was significantly lower with antibiotic treatment (8% vs. 20% in controls), thus resulting in comparable yields of dissoconch larvae ∼ 8% irrespective of treatment. A possible negative effect of antibiotic treatment on bacterial induction of settlement and metamorphosis of sea scallop larvae is suggested.

Settlement of larval blacklip abalone, Haliotis rubra, in response to green and red macroalgae

Surfaces from the habitat of adult Haliotis rubra were tested as inducers of larval settlement to determine the cues that larvae may respond to in the field. Settlement was high on the green algal species Ulva australis and Ulva compressa (Chlorophyta), the articulated coralline algae Amphiroa anceps and Corallina officinalis, and encrusting coralline algae (Rhodophyta). Biofilmed abiotic surfaces such as rocks, sand and shells did not induce settlement. Ulvella lens was also included as a control. Treatment of U. australis, A. anceps and C. officinalis with antibiotics to reduce bacterial films on the surface did not reduce the settlement response of H. rubra larvae. Similarly, treatment of these species and encrusting coralline algae with germanium dioxide to reduce diatom growth did not significantly reduce larval settlement. These results suggest that macroalgae, particularly green algal species, may play an important role in the recruitment of H. rubra larvae in the field and can be used to induce larval settlement in hatchery culture.

Induction of larval settlement in the polychaete Hydroides elegans by extracellular polymers of benthic diatoms

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.

Survival, growth, settlement and metamorphosis of refrigerated larvae of the mussel Mytilus galloprovincialis Lamarck and their use in settlement and antifouling bioassays

Straight-hinge veliger and pediveliger larvae of the mussel Mytilus galloprovincialis were refrigerated for varying periods for use in bioassays. Straight-hinge veliger larvae grew to the umbo-veliger stage after 2 months in the refrigerator, but no pediveligers were observed during the 3-month refrigeration period. The average survival rate of larvae in the refrigerator was 79% after 1 month, but gradually decreased with the refrigeration period, and was as low as 22% after 3 months. All refrigerated larvae grew to the pediveliger stage in the incubator at 17°C at the same rate as that of the control larvae that were not refrigerated. Settlement and metamorphosis of pediveligers from both refrigerated and control groups were facilitated by microbial film and epinephrine and inhibited by phentolamine. Thus, refrigeration can be used as an effective method of storing larvae of M. galloprovincialis for use in assays to assess candidate settlement inducers and antifouling substances.

Effects of GABA and epinephrine on the settlement and metamorphosis of the larvae of four species of bivalve molluscs

Competent larvae of different marine bivalve species were treated with GABA and epinephrine at different concentrations and times of exposure to test the ability of the drugs to induce settlement and metamorphosis. GABA induced both settlement and metamorphosis in the mussel Mytilus galloprovincialis, the clams Venerupis pullastra and Ruditapes philippinarum and the oyster Ostrea edulis. Maximum induction of settlement (>39%) was achieved after exposure of V. pullastra larvae to 10 −4 M GABA; this concentration of GABA also induced the highest percentages of metamorphosis in the four species studied. Epinephrine was identified as an active inducer of settlement and metamorphosis in bivalve molluscs. Exposure to 10 −5 M epinephrine induced significant levels of settlement in Mytilus, Venerupis and Ostrea. In contrast, epinephrine failed to induce settlement behaviour in Ruditapes. Maximum induction of metamorphosis was produced by 10 −5 M epinephrine in mussels, clams and oysters; Ruditapes showed the highest percentage of metamorphosis (>78%). This is the first report in which the involvement of GABA in the settlement and metamorphosis of bivalve molluscan larvae is demonstrated. It was also recognised that epinephrine plays a role not only in inducing metamorphosis but also in initiating settlement.

Induction of larval settlement and metamorphosis in the donkey-ear abalone, Haliotis asinina Linnaeus, by chemical cues

The effects of the chemical inducers, gamma-aminobutyric acid (GABA) and potassium chloride (KCl), on the larval settlement and metamorphosis of the donkey-ear abalone, Haliotis asinina, was investigated. H. asinina larvae (5–6 h post-hatch) were exposed to a range of GABA (0.125–2.00 μM) and KCl (1.00–12.00 mM) concentrations for 72 h. Results of the dose response experiments showed that settlement and metamorphosis vary according to the dose levels of the inducer compounds. Under controlled laboratory conditions, 0.45–0.50 μM and 6.0 mM seemed to be the optima for GABA and KCl, respectively, as these concentrations elicited the greatest number of postlarvae that metamorphosed, settled or survived. However, GABA generally promoted better attachment and metamorphic response as well as survival than KCl in H. asinina postlarvae.

Induction of larval settlement and metamorphosis of the abalone, Haliotis discus hannai larvae using bromomethane and potassium chloride

Larvae of Haliotis discus hannai were induced to settle and metamorphose by exposure to seawater supplemented with bromomethane. The settlement and metamorphosis of veliger larvae were observed with bromomethane concentrations of 300, 600, 900, 1200, 1500 ppm on the benthic diatom plates. The highest settlement and metamorphosis rates of larvae were observed in bromomethane concentrations of 600 ppm. Abalone larvae were induced to settle and metamorphose by exposure to seawater supplemented with potassium chloride. The settlement and metamorphosis of veliger larvae was observed in potassium chloride concentrations of 14, 19, 24 and 29 mM on the benthic diatom plates. The highest settlement and metamorphosis rates of larvae were observed in potassium chloride concentrations of 14-19 mM.

Induction of Larval Settlement in the Serpulid Polychaete Hydroides elegans (Haswell): Role of Bacterial Extracellular Polymers

Induction of Larval Settlement in the Serpulid Polychaete Hydroides elegans (Haswell): Role of Bacterial Extracellular Polymers

Induction of larval settlement in the serpulid polychaete Hydroides elegans (Haswell): Role of bacterial extracellular polymers

Larval settlement in the marine polychaete Hydroides elegans is effectively mediated upon contact with the surface of marine bacterial films. Using the bacterium Roseobacter litoralis as a model strain, the effect of bacterial extracellular polymers (exopolymers) on larval settlement of H. elegans was investigated. Bioassays with exopolymer fractions dissociated from bacterial films evoked the initial stages of the larval settlement process, i.e. larvae slowed down, secreted a mucous thread and crawled over the surface. This response is typical of larvae that encounter an attractive bacterial film. In contrast, bioassays with exopolymers in association with UV‐irradiated, metabolically inactive bacterial films evoked complete settlement. However, the percentage of responding larvae was negatively correlated with the magnitude of UV‐dosage. Since UV energy crosslinks both intra‐ and extracellular proteinaceous components, it could not be distinguished whether the decrease in larval settlement was due to a modification of proteinaceous components of exopolymers or due the elimination of cellular activity. Nevertheless, the results ascribe bacterial exopolymers the role of an indicator of substratum suitability and provide evidence that the polysaccharide moiety of exopolymers does not complement this effect.

Delayed metamorphosis in decapod crustaceans: evidence and consequences

Most marine invertebrate species exhibit a complex life cycle including a planktonic larval phase and a benthic juvenile-adult phase. Metamorphosis and settlement are the links between these phases of development. In many species, metamorphosis is triggered by specific chemical and/or physical cues, mainly associated with the adult habitat. In the absence of such cues. competent larvae can delay their metamorphosis by a few days to several months. Most investigations on the delay of metamorphosis have been realised on sessile or sedentary species. In relation to mobile decapod crustaceans. the number of such studies is low, probably because the members of this group retain their mobility after metamorphosis. and hence, may depend less on environmental cues for the induction of settlement and metamorphosis. Nevertheless. the larvae of some decapod species have been shown to depend on metamorphosis-stimulating cues. These include special types of substrates, physical or chemical traits of particular (e.g., estuarine) water bodies, as well as odors from conspecific or congeneric adults. The capacity for delay is, in the decapod species studied so far, limited and may normally end with spontaneous metamorphosis. An extended time of larval development presents the advantage of enhancing the probability for locating a suitable habitat, but it may imply, as a disadvantage, a reduction of juvenile growth or survival and a prolonged development time preceding benthic life. This paper reviews the available evidence for delayed metamorphosis in decapod crustaceans, indentifed cues, the importance of larval age at the time of contact with a cue, and costs of delayed metamorphosis. Additionally, we propose new frontiers for future investigations on delayed metamorphosis in decapod crustaceans, including the molecular identification of chemical cues, the identification of the stage(s) of the moulting cycle that is or are sensitive to such cues, the study of hormonally mediated effects on the moulting cycle, the quantification of energetic or other costs of delayed metamorphosis, and the analysis of relationships between the effectiveness of adult odors and phylogenetic proximity of larvae and adults.

Evaluation of mucus, Navicula, and mixed diatoms as larval settlement inducers for the tropical abalone Haliotis asinina

Abstract Different biological films reportedly effective in larval settlement of other abalone species were tested on the tropical abalone Haliotis asinina to determine the most effective settlement inducers for the species. Larval attachment was significantly higher on mucus, Navicula , Navicula +mucus, and mixed diatoms+mucus than on mixed diatoms and the blank control. Metamorphosis was significantly higher on Navicula , Navicula +mucus. These results indicate the important contribution of mucus in larval settlement and the effectiveness of Navicula as a single species over mixed diatoms in larval settlement of the tropical abalone H. asinina .

Pharmacological Induction of Larval Settlement and Metamorphosis in the Blue Mussel Mytilus edulis L.

The blue mussel Mytilus edulis L. is an important aquaculture and fouling species in northern seas. Although the general role of chemical cues for settlement of larvae of the blue mussel has been proposed, few studies have focused on induction of settlement and metamorphosis by pharmacological agents. In this study, the induction of larval settlement of the blue mussel by pharmacological compounds was investigated through a series of laboratory experiments with an aim of identifying artificial cues for laboratory bioassay systems in fouling and antifouling research. Gamma-aminobutiric acid (GABA), dihydroxyphenyl L-alanine (DOPA), isobutyl methylxanthine (IBMX) and acetylcholine chloride (ACH) at 10m 7-10m 2 M as well as KCl at 10-40 mM K+ in excess of the level in normal seawater were tested for their inductive effect on larval settlement. In filtered seawater (FSW) <9% of the larvae settled after 48 h. Elevated K+ and GABA levels had no effect on larval settlement and metamorphosis. DOPA at 10m 5 M and IBMX at 10m 6-10m 4 M induced 41-83% larval settlement and ACH at 10m 7-10m 5 M induced < 40% larval settlement. While the highest settlement rates were observed after 48 h exposure to the chemicals, most of the larvae settled within 24 h. Compounds at concentrations of 10m 3-10m 2 M were either toxic to larvae or retarded the growth of the post-larvae shell. Juveniles resulting from induction by lower concentrations of chemicals had a very high survival rate, completed metamorphosis and grew as well as the juveniles that metamorphosed spontaneously. IBMX at 10m 6-10m 4 M and L-DOPA at 10m 5 M are effective agents for induction of settlement and metamorphosis for future studies using juvenile M. edulis.

Induction of larval settlement in the polychaete Hydroides elegans by surface-associated settlement cues of marine benthic diatoms

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.