Surface-associated fucoxanthin mediates settlement of bacterial epiphytes on the rockweed Fucus vesiculosus

The chemical defence against microfouling in the brown seaweed Fucus vesiculosus was investigated and an inhibitor of bacterial settlement was isolated by bioassay-guided fractionation of non-polar surface extracts. UV-vis and mass spectrometry were used to identify the compound as the carotenoid fucoxanthin. The metabolite was tested at the natural concentration (in a surface volume based assay) against the settlement of four bacterial strains isolated from F. vesiculosus and 11 strains isolated from co-occurring algae and marine sediment. Surface concentrations between 1.4 and 6 μg cm^?2 resulted in 50% inhibition of four of these isolates, which were studied in more detail using a surface area-based assay, while a fifth isolate proved to be less sensitive. The presence of fucoxanthin on the surface of F. vesiculosus was demonstrated with two different surface extraction methods. Fucoxanthin was detected at concentrations between 0.7 and 9 μg cm^?2 on the algal surface. Fucoxanthin was still present at the algal surface after removal of associated diatoms through mechanical cleaning and germanium dioxide treatment and was thus mainly produced by F. vesiculosus rather than by diatoms. Thus, the photosynthetic pigment fucoxanthin appears to be ecologically relevant as a surface-associated antimicrobial agent, acting against the settlement of bacteria on the surface of the macroalga F. vesiculosus.     

Dimethylsulphopropionate (DMSP) and proline from the surface of the brown alga Fucus vesiculosus inhibit bacterial attachment

It was demonstrated previously that polar and non-polar surface extracts of the brown alga Fucus vesiculosus collected during winter from the Kiel Bight (Germany) inhibited bacterial attachment at natural concentrations. The present study describes the bioassay-guided identification of the active metabolites from the polar fraction. Chromatographic separation on a size-exclusion liquid chromatography column and bioassays identified an active fraction that was further investigated using nuclear magnetic resonance spectroscopy and mass spectrometry. This fraction contained the metabolites dimethylsulphopropionate (DMSP), proline and alanine. DMSP and proline caused the anti-attachment activity. The metabolites were further quantified on the algal surface together with its associated boundary layer. DMSP and proline were detected in the range 0.12-1.08 ng cm(-2) and 0.09-0.59 ng cm(-2), respectively. These metabolites were tested in the concentration range from 0.1 to 1000 ng cm(-2) against the attachment of five bacterial strains isolated from algae and sediment co-occurring with F. vesiculosus. The surface concentrations for 50% inhibition of attachment of these strains were always <0.38 ng cm(-2) for DMSP and in four cases <0.1 ng cm(-2) for proline, while one strain required 1.66 ng cm(-2) of proline for 50% inhibition. Two further bacterial strains that had been directly isolated from F. vesiculosus were also tested, but proved to be the least sensitive. This study shows that DMSP and proline have an ecologically relevant role as surface inhibitors against bacterial attachment on F. vesiculosus.     

高产岩藻黄素的海洋硅藻筛选及光照条件优化

为提高岩藻黄素(Fucoxanthin)提取效率, 对16株海洋硅藻岩藻黄素的检测含量和实际含量进行了分析, 并以一株牟氏角刺藻(Chaetoceros muelleri)为对象, 研究了岩藻黄素与另外两种关键光合色素(即叶绿素a和β-胡萝卜素)的含量及其消长受光强和光质影响的特征。研究结果显示, 不同种或同种不同株的海洋硅藻, 在相同的培养条件、收获时期、提取方法下, 其提取率差异大, 可从不足1%到89.78%; 岩藻黄素的检出含量各异, 从0.03到5.02 mg/g。受不同光照强度[低: 50 μmol/(m2·s); 中: 100 μmol/(m2·s); 高: 200 μmol/(m2·s)和光质(红光、蓝光)]的影响, 岩藻黄素产量呈现不同特征。在低光照强度下, 单位细胞岩藻黄素的含量相对较高。在红光条件下, 岩藻黄素的产量高于蓝光。在相同光强条件下, 岩藻黄素含量随增殖周期变化; 在单色光质条件下, 几种光合色素均在生长平台期后期含量增加。岩藻黄素的含量变化与叶绿素a和β-胡萝卜素的含量及其消长关系密切。研究为筛选藻株、优化硅藻培养条件以累积岩藻黄素提供了参考。     

Inhibition of common fouling organisms in mariculture by epiphytic bacteria from the surfaces of seaweeds and invertebrates

A total of 319 bacterial strains isolated from the surfaces of seaweeds and invertebrates were tested for their effects on settlement of Ulvalactuca spores and Hydroidesezoensis larvae in laboratory bioassays. Of the 192 bacterial strains isolated from the surfaces of seaweeds 63 isolates were shown to be inhibitory against the settlement of algal spores and 62 isolates were inhibitory against larval settlement. Thirty-seven percent of the 127 bacterial strains isolated from the surfaces of marine invertebrates were shown to be inhibitory against algal spores and larval settlement. The strain CI4 isolated from an adult Cionaintestinalis showed the strongest inhibitory effect and was identified as Pseudoalteromonas sp. via 16S rRNA gene sequencing. The high proportions of host associated bacteria producing antifouling compounds suggest that these bacteria may help the host organism in the defense against fouling.     

Inhibition of common fouling organisms in mariculture by epiphytic bacteria from the surfaces of seaweeds and invertebrates

A total of 319 bacterial strains isolated from the surfaces of seaweeds and invertebrates were tested for their effects on settlement of Ulvalactuca spores and Hydroidesezoensis larvae in laboratory bioassays. Of the 192 bacterial strains isolated from the surfaces of seaweeds 63 isolates were shown to be inhibitory against the settlement of algal spores and 62 isolates were inhibitory against larval settlement. Thirty-seven percent of the 127 bacterial strains isolated from the surfaces of marine invertebrates were shown to be inhibitory against algal spores and larval settlement. The strain CI4 isolated from an adult Cionaintestinalis showed the strongest inhibitory effect and was identified as Pseudoalteromonas sp. via 16S rRNA gene sequencing. The high proportions of host associated bacteria producing antifouling compounds suggest that these bacteria may help the host organism in the defense against fouling.     

Marine algal fucoxanthin inhibits the metastatic potential of cancer cells

Metastasis is major cause of malignant cancer-associated mortality. Fucoxanthin has effect on various pharmacological activities including anti-cancer activity. However, the inhibitory effect of fucoxanthin on cancer metastasis remains unclear. Here, we show that fucoxanthin isolated from brown alga Saccharina japonica has anti-metastatic activity. To check anti-metastatic properties of fucoxanthin, in vitro models including assays for invasion, migration, actin fiber organization and cancer cell–endothelial cell interaction were used. Fucoxanthin inhibited the expression and secretion of MMP-9 which plays a critical role in tumor invasion and migration, and also suppressed invasion of highly metastatic B16-F10 melanoma cells as evidenced by transwell invasion assay. In addition, fucoxanthin diminished the expressions of the cell surface glycoprotein CD44 and CXC chemokine receptor-4 (CXCR4) which play roles in migration, invasion and cancer–endothelial cell adhesion. Fucoxanthin markedly suppressed cell migration in wound healing assay and inhibited actin fiber formation. The adhesion of B16-F10 melanoma cells to the endothelial cells was significantly inhibited by fucoxanthin. Moreover, in experimental lung metastasis in vivo assay, fucoxanthin resulted in significant reduction of tumor nodules. Taken together, we demonstrate, for the first time, that fucoxanthin suppresses metastasis of highly metastatic B16-F10 melanoma cells in vitro and in vivo.     

Inhibition of common fouling organisms by marine bacterial isolates ith special reference to the role of pigmented bacteria

Two questions of relevance to the establishment of marine biofouling communities were addressed, viz (1) what is the frequency with which bacterial strains isolated from living and inanimate surfaces in the marine environment show inhibitory activity against the settlement of common fouling organisms, and (2) is the antifouling bacterium, D2, an inhabitant of different marine waters, and how unique is this bacterium, in its mode of action against different target organisms? With respect to the first question, ninety three marine bacteria isolated from various rock surfaces from the marine environment were tested against larvae of Balanus amphitrite and spores of Ulva lactuca. Settlement assays against the diatom Amphora sp. were also performed on 10 of these strains. Nine bacterial isolates were shown to be inhibitory against larval settlement and eight of these strains were also inhibitory against algal spores. Altogether 16 strains were inhibitory against the settlement of algal spores while none of the bacterial strains inhibited diatom settlement. With respect to the second question, D2, a dark green pigmented bacterium, isolated from an adult tunicate off the Swedish west coast, has been found to be a very effective inhibitor against common fouling organisms. In order to see if this bacterium can be found in other marine waters, bacteria from living surfaces of marine plants and animals from waters around Sydney, Australia, were isolated and screened for inhibitory activity against barnacle larvae. Seventy four percent of the 23 plant isolates were shown to be inhibitory against larval settlement while only 30% of the 23 isolates from marine animals reduced settlement. Twenty two of the isolates from different seaweeds were dark pigmented and 20 of these strains inhibited settlement of barnacle larvae and algal spores. Three of the strains showed the same phenotypic expression as D2, and the results indicate that these strains may be D2 or closely related strains, suggesting that D2 may be a common inhabitant in the marine environment.     

Defence Chemistry Modulation by Light and Temperature Shifts and the Resulting Effects on Associated Epibacteria of Fucus vesiculosus

The goals of this study were (1) to investigate whether Fucus vesiculosus regulates the production of its antifouling defence chemicals against epibacteria in response to light limitation and temperature shifts and (2) to investigate if different surface concentrations of defence compounds shape epibacterial communities. F. vesiculosus was incubated in indoor mesocosms at five different temperature conditions (5 to 25°C) and in outdoor mesocosms under six differently reduced sunlight conditions (0 to 100%), respectively. Algal surface concentrations of previously identified antifouling compounds - dimethylsulphopropionate (DMSP), fucoxanthin and proline – were determined and the bacterial community composition was characterized by in-depth sequencing of the 16S-rRNA gene. Altogether, the effect of different treatment levels upon defence compound concentrations was limited. Under all conditions DMSP alone appeared to be sufficiently concentrated to warrant for at least a partial inhibitory action against epibiotic bacteria of F. vesiculosus. In contrast, proline and fucoxanthin rarely reached the necessary concentration ranges for self-contained inhibition. Nonetheless, in both experiments along with the direct influence of temperature and light, all three compounds apparently affected the overall bacterial community composition associated with F. vesiculosus since tendencies for insensitivity towards all three compounds were observed among bacterial taxa that typically dominate those communities. Given that the concentrations of at least one of the compounds (in most cases DMSP) were always high enough to inhibit bacterial settlement, we conclude that the capacity of F. vesiculosus for such defence will hardly be compromised by shading or warming to temperatures up to 25°C.     

Disruption-free imaging by Raman spectroscopy reveals a chemical sphere with antifouling metabolites around macroalgae

Investigations of the surface chemistry of marine organisms are essential to understand their chemically mediated interactions with fouling organisms. In this context, the concentration of natural products in the immediate vicinity of algal surfaces, as well as their biological activity, are of particular importance. However, due to lack of appropriate methods, the distribution of compounds within the chemical sphere around marine algae is unknown. This study demonstrates the suitability of confocal resonance Raman microspectroscopy for the determination of metabolites around algal surfaces with a micrometer resolution. The spatial distribution of carotenoids in the diffusion boundary layer of the brown alga Fucus vesiculosus and the green alga Ulva sp. was determined using the disruption-free optical method. A gradient of carotenoids was determined within 0 to 150 μm from the surface of the algae, thereby demonstrating the release of the non-polar metabolites involved in antifouling processes. The differences in the carotenoid composition of the brown and green algae were reflected in the spectra. Resonance Raman microspectroscopy also allowed visualization of the lateral distribution of fucoxanthin on the algal surface and localization of concentration maxima within a 50 × 50 μm(2) area. The results from this work show clearly that established dipping techniques suitable for the extraction of the diffusion boundary layer of macroalgae only provide an average of the local strongly variable concentrations of metabolites on algal surfaces.     

A protective coat of microorganisms on macroalgae: inhibitory effects of bacterial biofilms and epibiotic microbial assemblages on barnacle attachment

Effects of epibiotic bacteria associated with macroalgae on barnacle larval attachment were investigated. Eight bacterial isolates obtained from samples of three macroalga species were cultured as monospecies bacterial films and tested for their activity against barnacle (Amphibalanus improvisus) attachment in field experiments (Western Baltic Sea). Furthermore, natural biofilm communities associated with the surface of the local brown alga, Fucus vesiculosus, which were exposed to different temperatures (5, 15 and 20?°C), were harvested and subsequently tested. Generally, monospecies bacterial biofilms, as well as natural microbial assemblages, inhibited barnacle attachment by 20-67%. denaturing gradient gel electrophoresis fingerprints showed that temperature treatment shifted the bacterial community composition and weakened the repellent effects at 20?°C. Repellent effects were absent when settlement pressure of cyprids was high. Nonviable bacteria tended to repel cyprids when compared to the unfilmed surfaces. We conclude that biofilms can have a repellent effect benefiting the host by preventing heavy fouling on its surface. However, severe settlement pressure, as well as stressful temperature, may reduce the protective effects of the alga's biofilm. Our results add to the notion that the performance of F.?vesiculosus may be reduced by multiple stressors in the course of global warming.     

Dimethylsulphopropionate (DMSP) and proline from the surface of the brown alga Fucus vesiculosus inhibit bacterial attachment

It was demonstrated previously that polar and non-polar surface extracts of the brown alga Fucus vesiculosus collected during winter from the Kiel Bight (Germany) inhibited bacterial attachment at natural concentrations. The present study describes the bioassay-guided identification of the active metabolites from the polar fraction. Chromatographic separation on a size-exclusion liquid chromatography column and bioassays identified an active fraction that was further investigated using nuclear magnetic resonance spectroscopy and mass spectrometry. This fraction contained the metabolites dimethylsulphopropionate (DMSP), proline and alanine. DMSP and proline caused the anti-attachment activity. The metabolites were further quantified on the algal surface together with its associated boundary layer. DMSP and proline were detected in the range 0.12–1.08 ng cm^?2 and 0.09–0.59 ng cm^?2, respectively. These metabolites were tested in the concentration range from 0.1 to 1000 ng cm^?2 against the attachment of five bacterial strains isolated from algae and sediment co-occurring with F. vesiculosus. The surface concentrations for 50% inhibition of attachment of these strains were always <0.38 ng cm^?2 for DMSP and in four cases <0.1 ng cm^?2 for proline, while one strain required 1.66 ng cm^?2 of proline for 50% inhibition. Two further bacterial strains that had been directly isolated from F. vesiculosus were also tested, but proved to be the least sensitive. This study shows that DMSP and proline have an ecologically relevant role as surface inhibitors against bacterial attachment on F. vesiculosus.     

Ecological role of a seaweed secondary metabolite for a colonizing bacterial community

Bacteria associated with seaweeds can both harm and benefit their hosts. Many seaweed species are known to produce compounds that inhibit growth of bacterial isolates, but the ecological role of seaweed metabolites for the associated bacterial community structure is not well understood. In this study the response of a colonizing bacterial community to the secondary metabolite (1,1,3,3-tetrabromo-2-heptanone) from the red alga Bonnemaisonia hamifera was investigated by using field panels coated with the metabolite at a range of concentrations covering those measured at the algal surface. The seaweed metabolite has previously been shown to have antibacterial effects. The metabolite significantly affected the natural fouling community by (i) altering the composition, (ii) altering the diversity by increasing the evenness and (iii) decreasing the density, as measured by terminal restriction fragment length polymorphism in conjunction with clone libraries of the 16S rRNA genes and by bacterial enumeration. No single major bacterial taxon (phylum, class) was particularly affected by the metabolite. Instead changes in community composition were observed at a more detailed phylogenetic level. This indicates a broad specificity of the seaweed metabolite against bacterial colonization, which is supported by the observation that the bacterial density was significantly affected at a lower concentration (0.02 μg cm?2) than the composition (1-2.5 μg cm?2) and the evenness (5 μg cm?2) of the bacterial communities. Altogether, the results emphasize the role of secondary metabolites for control of the density and structure of seaweed-associated bacterial communities.     

Antibacterial and antilarval activity of deep-sea bacteria from sediments of the West Pacific Ocean

Abstract

Deep-sea microorganisms are a new source of bioactive compounds. In this study, crude ethyl acetate extracts of 176 strains of deep-sea bacteria, isolated from sediments of the West Pacific Ocean, were screened for their antibacterial activity against four test bacterial strains isolated from marine biofilms. Of these, 28 deep-sea bacterial strains exhibited antibacterial activity against one or more of the bacteria tested. Active deep-sea bacterial strains belonged mainly to the genera of Pseudomonas, Psychrobacter and Halomonas. Additionally, antilarval activity of 56 deep-sea bacterial strains was screened using Balanus amphitrite larvae. Seven bacterial strains produced metabolites that had strong inhibitive effects on larval settlement. None of these metabolites showed significant toxicity. The crude extract of one deep-sea Streptomyces strain could completely inhibit larval settlement at a concentration of 25 μg ml^?1.     

Antibacterial and antilarval activity of deep-sea bacteria from sediments of the West Pacific Ocean

Deep-sea microorganisms are a new source of bioactive compounds. In this study, crude ethyl acetate extracts of 176 strains of deep-sea bacteria, isolated from sediments of the West Pacific Ocean, were screened for their antibacterial activity against four test bacterial strains isolated from marine biofilms. Of these, 28 deep-sea bacterial strains exhibited antibacterial activity against one or more of the bacteria tested. Active deep-sea bacterial strains belonged mainly to the genera of Pseudomonas, Psychrobacter and Halomonas. Additionally, antilarval activity of 56 deep-sea bacterial strains was screened using Balanus amphitrite larvae. Seven bacterial strains produced metabolites that had strong inhibitive effects on larval settlement. None of these metabolites showed significant toxicity. The crude extract of one deep-sea Streptomyces strain could completely inhibit larval settlement at a concentration of 25 microg ml(-1).     

EFFECTS OF IRON AND LIGHT STRESS ON THE BIOCHEMICAL COMPOSITION OF ANTARCTIC PHAEOCYSTIS SP. (PRYMNESIOPHYCEAE). II. PIGMENT COMPOSITION

A strain of Phaeocystis sp., isolated in the Southern Ocean, was cultured under iron- and light-limited conditions. The cellular content of chlorophyll a and accessory light-harvesting (LH) pigments increased under low light intensities. Iron limitation resulted in a decrease of all light-harvesting pigments. However, this decrease was greatly compensated for by a decrease in cell volume. Cellular concentrations of the LH pigments were similar for both iron-replete and iron-deplete cells. Concentrations of chlorophyll a were affected only under low light conditions, wherein concentrations were suppressed by iron limitation. Ratios of the LH pigments to chlorophyll a were highest for iron-deplete cells under both light conditions. The photoprotective cycle of diato/diadinoxanthin was activated under high light conditions, and enhanced by iron stress. The ratio of diatoxanthin to diadinoxanthin was highest under high light, low iron conditions.   Iron limitation induced synthesis of 19'-hexanoyloxyfucoxanthin and 19'-butanoyloxyfucoxanthin at the cost of fucoxanthin. Fucoxanthin formed the main carotenoid in iron-replete Phaeocystis cells, whereas for iron-deplete cells 19'-hexanoyloxyfucoxanthin was found to be the main carotenoid. This shift in carotenoid composition is of importance in view of the marker function of both pigments, especially in areas where Phaeocystis sp. and diatoms occur simultaneously. A hypothesis is presented to explain the transformation of fucoxanthin into 19'-hexanoyloxyfucoxanthin and 19'-butanoyloxyfucoxanthin, referring to their roles as a light-harvesting pigment.     

Antioxidant and pro-oxidant activities of the brown algae, Laminaria digitata, Himanthalia elongata, Fucus vesiculosus, Fucus serratus and Ascophyllum nodosum

The ability of Laminaria digitata, Himanthalia elongata, Fucus vesiculosus, Fucus serratus and Ascophyllum nodosum to scavenge peroxyl radicals was investigated by kinetic studies in a model system. The thermal initiated oxidation of methyl linoleate was performed at 60°C in heptanol, with or without antioxidants. When they reached 1% of the substrate, seaweed extracts exhibited antioxidant activities by extending the induction period, but they did not suppress the rate of oxygen uptake as did vitamin E. A synergistic effect occurred when both algal extracts (1.5 g L^-1) and vitamin E (0.4 mmol L^-1) were present, and the effectiveness of the combined antioxidants during the whole induction period was vitamin E effectiveness. The synergistic effect of L. digitata, however, was subject to seasonal variations: samples collected in summer were effective synergists, whereas samples collected in winter displayed a marked negative synergism. The phospholipid fractions of F. vesiculosus, F. serratus and A. nodosum, including pigments, accounted for only 6% of the total lipid fraction, and did not exhibit a large synergistic effect. The main phospholipid was not phosphatidyl ethanolamine as usually related, but phosphatidyl inositol. Fucoxanthin had some antioxidant activity per se under our experimental conditions, but did not act as a synergist of vitamin E. The most potent synergists were recognized as chlorophyll a and related compounds by the application of liquid-liquid partition and chromatography for the identification of active components. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolated thallus-associated compounds from the macroalga Fucus vesiculosus mediate bacterial surface colonization in the field similar to that on the natural alga

This study investigated whether surface-associated compounds isolated from the macroalga Fucus vesiculosus had the potential to mediate microbial and/or macrobial epibiosis similar to that on the natural alga. To selectively yield thallus-associated compounds and avoid contamination by intracellular algal compounds, cell lysis was monitored by surface microscopy of algal cells and chemical profiling of algal surface extracts by coupled gas chromatography mass spectroscopy. The optimized extraction resulted in polar and non-polar algal surface extracts. The non-polar surface extract was immobilized in hydrogel, the polar surface extract was homogeneously perfused through the gel to ensure a temporally constant delivery of polar extract components. During a 7 day field trial, bacterial biofilms were formed on control gels and gels featuring polar and/or non-polar extract components. PERMANOVA revealed that bacterial community profiles on controls and on gels featuring polar or non-polar extract were significantly different from the profile on F. vesiculosus, while the profile on the gels bearing both polar and non-polar extracts was not. Moreover, the polar surface extracts inhibited the settlement of barnacle cyprids. Considering the pronounced effects of bacterial biofilms on invertebrate larval settlement, these results suggest that algal surface chemistry affects macrofouling not only directly but also indirectly, via its control of biofilm formation and composition.     

Fucoxanthin Inhibits the Inflammation Response in Paw Edema Model through Suppressing MAPKs,Akt, and NFκB

Undaria pinnatifida is a well‐known traditional Korean food with a variety of biological activities. Carrageenan (carr) is commonly used to induce paw edema in animal models. This study was designed to elucidate the processes underlying the anti‐inflammatory effect of fucoxanthin isolated from the sporophyll of U. pinnatifida in carr‐induced paw edema in ICR mice. Fucoxanthin significantly decreased carr‐induced increased nitric oxide levels in the plasma of mice with carr‐induced paw edema. Fucoxanthin protected catalase (CAT) and superoxide dismutase (SOD) activity against disruption in mice with carr‐induced paw edema. In addition, fucoxanthin repressed carr‐induced activation of inducible nitric oxide synthase, cyclooxygenase‐2, and nuclear factor kappa B, as well as carr‐induced phosphorylation of mitogen‐activated protein kinase, extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase, p38, and protein kinase B/Akt. These results suggest that fucoxanthin may have therapeutic potential as a treatment for patients with inflammatory diseases.     

Inhibitory effect of phenolic compounds and marine bacteria on larval settlement of the barnacle Balanus amphitrite amphitrite darwin

This study examined the inhibitory effect of 3 phenolic compounds and 12 strains of marine bacteria on the larval settlement of Balanus amphitrite amphitrite. The phenolic compounds used were phlorotannins, phloroglucinol and tannic acid. Phlorotannins are polymers of phloroglucinol (1,3,5‐trihydroxybenzene) known only from brown algae. Tannic acid, which exists in terrestrial plants, is composed of oligomers of phloroglucinol attached to a sugar molecule. The bacterial strains used were isolated from a natural biofilm. The following were investigated: 1) the toxicity of the phenolic compounds to B. a. amphirite in three different larval stages, viz. nauplius II, nauplius V and cyprid; 2) the potency of the compounds as inhibitors of larval settlement and the possible mechanism involved in settlement inhibition; and 3) the effects of the bacteria on larval settlement. The level of toxicity of the phenolic compounds varied widely for the larvae. Phlorotannins were most toxic, having LC₅₀ values ranging from 9.47 to 40.35 μg ml^‐1; phloroglucinol was least toxic, having LC₅₀ values of 235.12 to 368.28 μg ml^‐1. In general, nauplii were more sensitive to the toxicity of the phenolic compounds than cyprids. The greater sensitivity of nauplii may be due to their active feeding behavior, which exposes the interior of their bodies to the compounds by active intake. Phloroglucinol was the most potent settlement inhibitor, having an EC₅₀ value of 0.02 μg ml^‐1. Phlorotannins and tannic acid had EC₅₀ values of 1.90 μg ml^‐1 and 14.05 μg ml^‐1, respectively. Phloroglucinol appeared to inhibit larval settlement through a relatively non‐toxic mechanism as its LC₅₀ value was four orders of magnitude higher than its EC₅₀ value. The high potency of phloroglucinol indicates that a simple constituent of a complex natural compound can be more effective than the natural compound itself. Larval settlement bioassays with monospecies bacterial films indicated that some of the bacterial species were inhibitory to larval settlement while the others showed no effect. None of the bacterial strains in this study induced larval settlement.