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.     

Fouling mediates grazing: intertwining of resistances to multiple enemies in the brown alga Fucus vesiculosus

Macroalgae have to cope with multiple natural enemies, such as herbivores and epibionts. As these are harmful for the host, the host is expected to show resistance to them. Evolution of resistance is complicated by the interactions among the enemies and the genetic correlations among resistances to different enemies. Here, we explored genetic variation in resistance to epibiosis and herbivory in the brown alga Fucus vesiculosus, both under conditions where the enemies coexisted and where they were isolated. F. vesiculosus showed substantial genetic variation in the resistance to both epibiosis and grazing. Grazing pressure on the alga was generally lower in the presence than in the absence of epibiota. Furthermore, epibiosis modified the susceptibility of different algal genotypes to grazing. Resistances to epibiosis and grazing were independent when measured separately for both enemies but positively correlated when both these enemies coexisted. Thus, when the enemies coexisted, the fate of genotypes with respect to these enemies was intertwined. Genotypic correlation between phlorotannins, brown-algal phenolic secondary metabolites, and the amount of epibiota was negative, indicating that these compounds contribute to resistance to epibiosis. In addition, phlorotannins correlated also with the resistance to grazing, but this correlation disappeared when grazing occurred in the absence of epibiota. This indicates that the patterns of selection for the type of the resistance as well as for the resistance traits vary with the occurrence patterns of the enemies.     

Resistance of the brown alga Fucus vesiculosus to herbivory

Herbivory is typically intense in marine littoral environments; thus, macrophytes are expected to evolve defenses against grazing. Although putative defenses of macrophytes are widely studied, there is lack of studies demonstrating the main premises of defense adaptations: the consequences of herbivory to macrophytes, genetic variation of defense traits and the costs and benefits of defenses in natural environment. We conducted a factorial experiment, where we manipulated amount of herbivory, growing depth and nutrient availability, and measured resistance to herbivory as well as genetic variation and costs of phlorotannins, putative defensive secondary metabolites, in the brown alga Fucus vesiculosus . Herbivory on algae varied with depth: grazing did not cause losses close to the surface, but, most of the algal production was consumed at the deeper end of the algal belt. The higher the genotypic phlorotannin content the less damage the genotype received implying that phlorotannins acted as a resistance trait. Production of phlorotannins was associated with costs for growth. Consistent with the prediction that the cost of defense will be greatest when resources are limiting, the cost appeared only in the deep end of the algal belt where growth was slowed down. Phlorotannins displayed phenotypic plasticity; the three factors influenced phlorotannins interactively, with the main tendencies of nutrient enrichment decreasing and herbivory and increasing depth increasing phlorotannins. Despite this plasticity, variation of phlorotannins was mainly due to the genotype of algae. These results emphasize the role of herbivory as a selective agent for algal defenses and the importance of genetic variation in the constitutive level of phlorotannins in interactions with natural enemies. The cost of phlorotannins may constrain the evolution of resistance in environments where growth is limited by light availability.     

Dielectric-Gravimetric Measurements on Fucus vesiculosus and Ascophyllum nodosum During Dehydration

Careri, G., Giansanti, A. and Fensom, D. 1986. Dielectnc-gravimetricmeasurements on Fucus vesiculosus and Ascophyllum nodosum duringdehydration.—J. exp. Bot. 37: 375–378. Samples of the marine algae Fucus vesiculosus and Ascophyllumnodosum were dehydrated between plates of a condenser so thatsimultaneous measurements of dielectric properties and rateof water loss could be made. During the desiccation process,both the evaporation rate and the dielectric properties becomezero at a critical hydration H_c, indicating that for H <H_c,the residual water must be in a tightly bound state. The absolutevalues of H_c, depend on the nature of the sample, but they arein the range 0·1–0·3, well above the hydrationlimit for active ion uptake (H=0·02). It is concludedthat, in these fucoids, living cells are maintained in the hydrationregion where water is in the bound state only. Key words: Fucus, Ascophyllwn, dielectric, dehydration     

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.     

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.     

The Composition of the Gas contained in the Vesicles of Fucus vesiculosus

Analysis of the gases contained in the vesicles of Fucus vesiculosusshowed that their oxygen content may be markedly enhanced byphotosynthesis. Under conditions of exposure to the air it was found that theoxygen content rarely fell much below 20 per cent. even afterprolonged periods in the dark. The carbon dioxide content of the vesicular gas varied from0 to 2·5 per cent. and appeared to bear no relationshipto metabolic activity. The results as a whole suggested that vesicle formation maypossibly be conditioned by the intensity of photosynthetic activity.     

High-performance liquid chromatographic analysis of phlorotannins from the brown alga Fucus vesiculosus

Separating individual compounds by HPLC represents an effective method for the detection and quantification of phenolic compounds and has been widely utilised. However, phlorotannins are commonly quantified using colorimetric methods, as the total amount of the whole compound group. In the present paper the separation of a set of individual soluble phlorotannins from the phenolic crude extract of Fucus vesiculosus was achieved by HPLC with UV photodiode array detection. Different gradient programs for reversed- and normal-phase HPLC methods were developed and tested. Normal-phase (NP) conditions with a silica stationary phase and a mobile phase with a linear gradient of increasing polarity were found to separate 16 individual components of the phenolic extract. The suitability of the NP-HPLC method for mass spectrometric application was preliminarily tested. Sample preparation was found to be a critical step in the analysis owing to the rapid oxidation of phlorotannins; ascorbic acid was used as an antioxidant.     

Geographical variation in Fucus vesiculosus morphology in the Baltic and North Seas

Effects of stress and disturbance on morphology, reproductive effort, size and sex ratio were studied for Fucus vesiculosus populations from the Baltic Sea at Askö and the North Sea on the west coast of Sweden at Tjäm[otilde]. High morphological variation was found between Fucus populations, with significant differences in length and weight of individuals, thallus breadth, number of branches and receptacles and receptacle weight, not only between Baltic and North Sea populations but also between populations within the same area, differing in wave exposure. With increasing disturbance, individuals in both studied populations were smaller and less branched. Differences were observed in plant size, with longer, broader and more branched plants being found in Askö compared with Tjärnö. Fucus populations at Tjämö allocated more biomass to reproduction and had longer, heavier receptacles than at Askö. Although the observed morphological changes may be partly explained by differences in wave exposure and salinity between the two sites, it is not possible to rule out genetic differences between the Baltic and North Sea populations. However, it is unlikely that the variations observed within the populations and between populations from the same area are genetically determined.     

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.     

Intriguing asexual life in marginal populations of the brown seaweed Fucus vesiculosus

Reproduction of attached large brown algae is known to occur only by sexual zygotes. Using microsatellites we show evolution of asexual reproduction in the bladder wrack promoting population persistence in the brackish water Baltic Sea (< 6 psu). Here a dwarf morph of Fucus vesiculosus is dominated by a single clone but clonal reproduction is also present in the common form of the species. We describe a possible mechanism for vegetative reproduction of attached algae, and conclude that clonality plays an important role in persistence and dispersal of these marginal populations, in which sexual reproduction is impaired by low salinity.     

Role of Auxin in Induction of Polarity in Fucus vesiculosus Zygotes

We studied the effects of auxin (indole-3-acetic acid) on formation of the primary polarity axis in zygotes of the brown algae Fucus vesiculosusL. Within the first 2.5 h after fertilization, the zygotes release this phytohormone in the ambient medium. The treatment of developing zygotes with the inhibitor of indole-3-acetic acid transport from the cell 2,3,5-triiodobenzoic acid at 5 mg/l arrests the auxin secretion and leads to its accumulation in the cells. This causes a significant delay in zygote polarization. The treatment of zygotes with the exogenous indole-3-acetic acid at 1 mg/l stimulates cell polarization and formation of a rhizoid protuberance. When auxin was added to the medium with triiodobenzoic acid, the inhibitory effect of the latter was eliminated. It has been proposed that the content of indole-3-acetic acid in the ambient medium is a key factor in the induction of polarity of the F. vesiculosus zygotes.     

Extraction of sulfated polysaccharides by autohydrolysis of brown seaweed Fucus vesiculosus

The extraction of sulfated polysaccharides (fucoidan) by autohydrolysis (AH) of brown seaweed Fucus vesiculosus was studied. Experimental assays were performed under different conditions of temperature (160 to 200°C) and reaction time (10 to 30 min) according to a 2² central composite design, and the conditions able to maximize the fucoidan yield were selected. The alga degradation and the total sugar yield in the liquor after AH were also determined to each experimental condition. The highest fucoidan yield (～16.5% w/w) was obtained when the AH process was performed at 180°C for 20 min. This product was characterized by high-performance liquid chromatography, infrared analysis spectroscopy, and thermal gravimetric analyses, which verified the presence of fucose and galactose as main components (70:30% mol ratio, in average) and an SO₃ content higher than 20%. AH process under optimum reaction conditions was an effective method to recover fucoidan from F. vesiculosus. The use of this technology brings also important advantages from economical and environmental viewpoints since it does not require the use of chemical solvent and generates less waste when compared to conventional extraction procedures.     

Seasonal variation in the antifouling defence of the temperate brown alga Fucus vesiculosus

The important role of marine epibiotic biofilms in the interactions of the host with its environment has been acknowledged recently. Previous studies with the temperate brown macroalga Fucus vesiculosus have identified polar and non-polar compounds recovered from the algal surface that have the potential to control such biofilms. Furthermore, both the fouling pressure and the composition of the epibiotic bacterial communities on this macroalga varied seasonally. The extent to which this reflects a seasonal fluctuation of the fouling control mechanisms of the host is, however, unexplored in an ecological context. The present study investigated seasonal variation in the anti-settlement activity of surface extracts of F. vesiculosus against eight biofilm-forming bacteria isolated from rockweed-dominated habitats, including replication of two populations from two geographically distant sites. The anti-settlement activity at both sites was found to vary temporally, reaching a peak in summer/autumn. Anti-settlement activity also showed a consistent and strong difference between sites throughout the year. This study is the first to report temporal variation of antifouling defence originating from ecologically relevant surface-associated compounds.     

Lipid Metabolism in the Brown Marine Algae Fucus vesiculosus and Ascophyllum nodosum

Lipid metabolism and environmental effects on this process havebeen studied in the marine brown algae Fucus vesiculosus andAscophyllum nodosum. These algae showed very similar patternsof lipid metabolism during 24 h incubations. Labelling from[1-¹⁴C]acetate showed the major labelled lipids to be the ß-alanineether lipid and the neutral lipid fraction in both algae. Ofthe glycolipids, only sulphoquinovosyldiacylglycerol was welllabelled and the phosphoglycerides were all poorly labelled.The major labelled fatty acids were palmitate and oleate, againin both algae, although Fucus vesiculosus also showed significantlabelling of stearate and behenate. Although the amount of fattyacid labelling increased with time, the proportion of labelin palmitate and oleate remained approximately constant. Verylong chain fatty acids (arachidic, behenic) were increasinglylabelled with time. Lowered incubation temperatures decreased labelling of the saturatedfatty acids. Cu²⁺ increased the proportion of oleate labelledin both algae, and of linoleate in Fucus vesiculosus. This cationdecreased the percentage labelling of stearate and myristatein Ascophyllum nodosum. Lipid metabolism in Ascophyllum nodosumwas more sensitive to raised Cu²⁺ levels than in Fucus vesiculosus Key words: Acyl lipid metabolism, Fucus vesiculosus, temperature effects, Ascophyllum nodosum, copper pollution     

Photosynthesis and UV-B Tolerance of the Marine Alga Fucus vesiculosus at Different Sea Water Salinities

The marine algal species in the Baltic Sea are few due to the low sea water salinity. One of the few species that can be found is Fucus vesiculosus. Even this species is affected by the low salinity and becomes smaller in size in the Baltic. In present work the photosynthesis of F. vesiculosus in the northern Baltic (Bothnian Sea) was compared to the photosynthesis of F. vesiculosus in the Atlantic. Oxygen evolution was measured before and after exposure to 2.3 W of UV-B (280–320 nm) radiation for 5 h, as well as after 48 h recovery in low light. The plants were kept in their own sea water salinity as well as in a changed salinity, this to examine possible correlations between salinity and photosynthesis. The results show a significant higher initial maximal photosynthesis (P _max) for Atlantic plants (10.3 nmol O₂ g⁻¹ FW s⁻¹) compared to Baltic plants (4.0 nmol O₂ g⁻¹ FW s⁻¹). The Baltic plants were found more sensitive to UV-B with a 40–50% decrease of P _max as well as a lower degree of recovery (60–70% compared to 75–95% for the Atlantic plants). The higher salinity (35 psu) had a positive effect on the Baltic F. vesiculosus with increased P _max as well as increased tolerance to UV-B. The lower salinity (5 psu) had a negative effect on the Atlantic plants with a decreased P _max as well as a lower tolerance to UV-B. Pigment content was found higher in Atlantic F. vesiculosus. The pigment content decreased then the Atlantic plants were transferred to 5 psu. The concentration of Chl a as well as the total content of violaxanthin, diadinoxanthin and zeaxanthin in Baltic plants increased when transferred to 35 psu. The Atlantic F. vesiculosus can not survive the low salinity in the northern Baltic (died within 8 weeks). It is likely that a long time acclimation or adaptation to low salinity has taken place for F. vesiculosus in northern Baltic. If this is an ecotypic or genotypic development it is too early to say.     

Variation in natural selection for growth and phlorotannins in the brown alga Fucus vesiculosus

Directional selection for plant traits associated with resistance to herbivory tends to eliminate genetic variation in such traits. On the other hand, balancing selection arising from trade-offs between resistance and growth or spatially variable selection acts against the elimination of genetic variation. We explore both the amount of genetic variation and variability of natural selection for growth and concentration of phenolic secondary compounds, phlorotannins, in the brown alga Fucus vesiculosus. We measured variation in selection at two growing depths and two levels of nutrient availability in algae that had faced two kinds of past growing environments. Genetic variation was low for growth but high for phlorotannins. The form and strength of selection for both focal traits depended on the past growing environment of the algae. We found strong directional selection for growth rate in algae previously subjected to higher ultraviolet radiation, but not in algae previously subjected to higher nutrient availability. Stabilizing selection for growth occurred especially in the deep growing environment. Selection for phlorotannins was generally weak, but in some past-environment-current-environment combinations we detected either directional selection against phlorotannins or stabilizing selection. Thus, phlorotannins are not selectively neutral but affect the fitness of F. vesiculosus. In particular, there may be a fitness cost of producing phlorotannins, but the realization of such a cost varies from one environment to another. Genetic correlations between selective environments were high for growth but nonexistent for phlorotannins, emphasizing the high phenotypic plasticity of phlorotannin production. The highly heterogeneous selection, including directional, stabilizing, and spatially variable selection as well as temporal change in selection due to responses to past environmental conditions, probably maintains a high amount of genetic variation in phlorotannins. Such variation provides the potential for rapid evolutionary response of phlorotannins under directional selection.