THE FUCUS (PHAEOPHYCEAE) SPERM RECEPTOR FOR EGGS. II. ISOLATION OF A BINDING PROTEIN WHICH PARTIALLY ACTIVATES EGGS1

An in vitro binding assay involving egg plasma membrane vesicles (PMVs) of Fucus serratus L. and proteins contained in a KCl extract of sperm has been used to identify a sperm protein involved in egg binding. High-performance gel filtration (HPGF) separated the sperm KCl extract into several major fractions, and a protein (apparent M, 60 kDa) was identified as being involved in binding to the egg PMVs. This protein ran on denaturing sodium dodecyl sulfate (SDS)gels with an apparent molecular weight of 27 kDa. This suggests that either the native form of the protein is a dimer or the molecular weight on HPGF is an artifact caused by high ionic strength buffer promoting hydrophobic interactions. When KCl-sol-uble proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE), blotted onto nitrocellulose, and incubated with biotinylated egg PMVs, these bound to a band at 27 kDa, confirming the role of this protein. Addition of the Fucus sperm extract or HPGF fractions containing the binding protein to eggs in the absence of sperm induced the release of polysaccharides onto the egg cell surface. This labeling was patchy, in contrast to the uniform release of polysaccharides observed when sperm were added to eggs. The monoclonal antibody (MAb) FS17 was raised against the 27-kDa sperm protein. It labeled the sperm body and both flagella by immunofluorescence, though the sperm had to he permeabilized to observe labeling, suggesting that the epitope recognized is not exposed at the cell surface. Addition of FS17 to the KCl extract in the binding assay reduced subsequent binding of egg PMVs. Removal of the 27-kDa protein recognized by FS17 from the sperm extract prevented the binding of egg PMVs in the binding assay and the triggering of the patchy release of polysaccharides when added to eggs. Overall the results suggest that the 27-kDa sperm protein is involved in binding to the egg plasma membrane and can trigger partial activation of the egg .     

IN VIVO MEASUREMENT OF ACTIVE OXYGEN PRODUCTION IN THE BROWN ALGA FUCUS EVANESCENS USING 2′,7′-DICHLOROHYDROFLUORESCEIN DIACETATE1

Intracellular production of active oxygen in the brown alga Fucus evanescens C. Ag. was studied by measuring the capacity for in vivo conversion of 2′,7′-dichlorohydrofluorescein diacetate (DCFH-DA) to the fluorescent dye 2′,7′-dichlorofluorescein (DCF), both in emersed and immersed seaweeds. Algae were incubated in seawater containing DCFH-DA under a range of conditions, and it was also possible to load algae with DCFH-DA and then follow subsequent DCF production in emersed tissue. DCF formation was linear for at least 2 h in both darkness and light, with the rate of formation increasing with the light level. DCF formation was temperature dependent. It also increased when algae were treated with H₂O₂ or methyl viologen (paraquat), which disrupts photosystem 1 electron transport and increases O^?₂ production. Exogenous catalase reduced in vivo DCF production, presumably by lowering cellular concentrations of H₂O₂. Hydrogen peroxide was released into the seawater by illuminated algae resulting in external dye conversion to DCF. However, this does not interfere with in vivo measurement of DCF by loaded, washed algae because DCF leakage appeared to be negligible. Internal DCF did not affect photosynthetic oxygen production relative to untreated controls. Overall, our data suggest that DCFH-DA is a potentially very useful probe for studying active oxygen metabolism in seaweeds subjected to environmental stresses.     

Littorina mariae — a factor structuring low shore communities?

The role of littorinids in structuring communities is discussed. On hard substrates preferential grazing of the dominant algae often enables competitively inferior species to utilize the rock substrate. Investigations of these systems has led to the proposal of a number of factors that should be analyzed in order to assess the effect of herbivores on community structure. Some of these factors have been investigated for Littorina mariae Sacchi & Rastelli. L. mariae is a micro-epiphytic grazer browsing the surface covering of epiphytes off the alga Fucus serratus (L.). The relationship between the host alga, F. serratus, and L. mariae is far more intricate than that between winkles and hard substrates as the alga itself is a dynamic resource. L. mariae is spatially and temporally linked to F. serratus. L. mariae is found almost exclusively on the alga and is positively attracted by extracts of the alga. The life history of the winkle is closely synchronized with that of the alga (the winter decrease of L. mariae populations is associated with the seasonal die-back of the host alga). On analysis of the factors considered important to assess herbivory, and examples of the effects of other epiphytic herbivores on algal success, it is suggested that L. mariae could potentially play an important role in structuring the community of it's host fucoid and that this may influence larger scale community structure. Experimental manipulations are proposed to evaluate these hypotheses.     

Parallel speciation or long‐distance dispersal? Lessons from seaweeds (Fucus) in the Baltic Sea

Parallel evolution has been invoked as a forceful mechanism of ecotype and species formation in many animal taxa. However, parallelism may be difficult to separate from recently monophyletically diverged species that are likely to show complex genetic relationships as a result of considerable shared ancestral variation and secondary hybridization in local areas. Thus, species' degrees of reproductive isolation, barriers to dispersal and, in particular, limited capacities for long‐distance dispersal will affect demographical structures underlying mechanisms of divergent evolution. Here, we used nine microsatellite DNA markers to study intra‐ and interspecific genetic diversity of two recently diverged species of brown macroalgae, Fucus radicans (L. Bergström & L. Kautsky) and F. vesiculosus (Linnaeus), in the Baltic Sea. We further performed biophysical modelling to identify likely connectivity patterns influencing the species' genetic structures. For each species, we found intraspecific contrasting patterns of clonality incidence and population structure. In addition, strong genetic differentiation between the two species within each locality supported the existence of two distinct evolutionary lineages (F_ST = 0.15–0.41). However, overall genetic clustering analyses across both species' populations revealed that all populations from one region (Estonia) were more genetically similar to each other than to their own taxon from the other two regions (Sweden and Finland). Our data support a hypothesis of parallel speciation. Alternatively, Estonia may be the ancestral source of both species, but is presently isolated by oceanographic barriers to dispersal. Thus, a limited gene flow in combination with genetic drift could have shaped the seemingly parallel structure.     

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.     

Polychlorinated biphenyls disrupt cell division and tip growth in two species of fucoid algae

Environmental contaminants, including poly‐chlorinated biphenyls (PCBs), are enriched in coastal sediments, and despite a 1977 moratorium by the United States Environmental Protection Agency on the production of PCBs, levels remain high, more so near former industrial plants. The effects of these contaminants on sessile species in the intertidal zone, particularly nonanimal species such as the ubiquitous fucoid brown algae, are not well known. We investigated the developmental effects of chronic PCB treatment beginning at fertilization on two species of marine rockweed, Fucus vesiculosus Linnaeus and Silvetia compressa (J.Agardh) E.Serrão, T.O.Cho, S.M.Boo & Brawley. A mixture of the most widely used PCB congeners, Aroclors 1221, 1242, and 1254, was delivered at concentrations well below levels found in contaminated sediments, and resulted in severely delayed mitosis and cytokinesis in both species. In F. vesiculosus, this delay was accompanied by abnormal spindle morphology. PCB treatment also dramatically slowed or arrested rhizoid growth after 2–4 d, and by 7 d F. vesiculosus embryos were dead; in contrast, polar secretion of adhesive, germination, and photopolar germination were not affected. The dramatic delay in the first cell division and reduction in tip growth within the first week of development are likely to compromise S. compressa's ability to reproduce and establish new generations. Thus, the data presented here suggest that PCBs still present in coastal sediments may be inhibiting recruitment in these species. Moreover, as sediment dredging causes temporary spikes in PCB concentrations, these kinds of bioremediation steps may exacerbate the disruption of fucoid development.     

Book review

Fucoid macroalgae are important primary producers and habitat modifiers on North Atlantic intertidal rocky shores. With decreasing latitude, western European fucoid populations display reduced levels of abundance, biomass and recruitment, while experiencing higher levels of physical environmental stress during summer months. We hypothesized that such reduction in the south is accompanied by a detectable decline in fucoid reproductive capacity. To test this hypothesis, morphological and reproductive traits of core (Welsh) and marginal (Portuguese) populations of two common fucoid species, Fucus vesiculosus and F. spiralis (Ochrophyta, Fucales), were examined. Morphological measurements showed that for a given thallus length, both fucoid species had smaller thallus volume and lower biomass in the southerly marginal part of the range. Significantly lower biomass of reproductive tissue of F. vesiculosus and a smaller number of receptacles per individual on specimens of both species indicate that levels of reproductive output are probably lower in southern populations. Despite the differences in reproductive traits observed between regions, reproductive effort (measured as the percentage of total dry biomass represented by reproductive tissue) of both species remained similar, as algae from both regions made similar investments in reproduction. The results indicate that stressful conditions reduced growth and number of receptacles of both species and amount of reproductive biomass of F. vesiculosus in the south but do not seem to change the way these algal species invest their energy. The decline in mass and reproductive biomass of specimens from southern shores found in this study, when combined with the lower abundance of adults and lower recruitment levels previously observed, is a strong indication of fucoid populations with lower levels of propagule output. This is an important factor when considering responses of these populations to a changing environment.     

Inheritance patterns of ITS1, chloroplasts and mitochondria in artificial hybrids of the seaweeds Fucus serratus and F. evanescens (Phaeophyceae)

Patterns of nuclear and organelle inheritance among artificial hybrids of the seaweeds Fucus serratus and F. evanescens were detected using single-strand conformation polymorphism (SSCP). Three alleles were identified in the 231?bp rDNA-ITS1 gene (nuclear): two in F. serratus and one in F. evanescens. Alleles differed by 1–2?bp and all hybrids possessed one allele from each parent. Two haplotypes were present in the 288?bp Rubisco spacer (chloroplast), differentiated by a 33?bp indel. Two haplotypes differing by a single nucleotide were found in a 135?bp region of nad11 gene (mitochondrion). Both organelles are maternally inherited, as all hybrids contained the haplotypes of the parent contributing the egg. Although laboratory hybrids among Fucus spp. have been produced previously, this is the first time that both nuclear and cytoplasmic genetic markers have been used to document inheritance patterns. SSCPs analysed on an automated sequencer offer a rapid and powerful approach for identifying suspected hybrids from field samples, as well as a screen for intraspecific and intra-individual variation in DNA regions prior to confirmation of variations by sequencing.     

Induction and genetic identification of a callus‐like growth developed in the brown alga Fucus vesiculosus

Induction of an axenic filamentous‐like callus growth from the brown algae Fucus vesiculosus is described. Different treatments were investigated in various combinations to develop axenic cultures based on identification of surface symbionts via 18S ribosomal RNA. Moreover, viability was confirmed after such processes by 2,3,5‐triphenyl tetrazolium chloride assay that demonstrated an average viability of 29%, relative to nonsterilized explants. After six weeks of a phototrophic cultivation on artificial sea water‐12‐nitrilotriacetic acid (0.5% w/v agar), a filamentous‐like callus growth was observed, which was identified genetically through its mitochondrial DNA after subculturing. Achievement of confirmed marine callus cultures might enrich old previously established blue biotechnology techniques and open new chances for cultivation of brown algae for production of good manufacturing practice‐compliant bioproducts.