Isolation of protoplasts and ion channel recording of plasma membrane patches and whole-cell recordings of the marine alga, <Emphasis Type="Italic">Ulva pertusa</Emphasis> (Chlorophyta)

Whole-cell patch-clamp techniques were used to study ion channels of a marine alga. High quality protoplasts suitable for electrophysiological studies were isolated from the green marine alga, Ulva pertusa, using enzyme mixtures consisting of cellulase and abalone power and identified by calcofluor fluorescence. The vitality of protoplasts varied depending on the alga growth stage, and those isolated from younger tissue in March maintained a high vitality with high sealing success rate compared with protoplasts isolated from mature or non-growing plants in August or November. In the whole-cell configuration, large inward currents were elicited by negative voltage pulses. The voltage-dependent component was predominantly carried by Cl⁻, as confirmed by the use of the Cl⁻ channel inhibitor DIDS and reversal potential of current-voltage plots. This evidence suggests that hyperpolarization-activated Cl⁻ permeable channels are responsible for the influx of Cl⁻ into U. pertusa cells. Voltage-dependent outward currents were also recorded in several protoplasts, and their properties need further investigation.     

A novel ether-linked phytol-containing digalactosylglycerolipid in the marine green alga, Ulva pertusa

Galactosylglycerolipids (GGLs) and chlorophyll are characteristic components of chloroplast in photosynthetic organisms. Although chlorophyll is anchored to the thylakoid membrane by phytol (tetramethylhexadecenol), this isoprenoid alcohol has never been found as a constituent of GGLs. We here described a novel GGL, in which phytol was linked to the glycerol backbone via an ether linkage. This unique GGL was identified as an Alkaline-resistant and Endogalactosylceramidase (EGALC)-sensitive GlycoLipid (AEGL) in the marine green alga, Ulva pertusa. EGALC is an enzyme that is specific to the R-Galα/β1-6Galβ1-structure of galactolipids. The structure of U. pertusa AEGL was determined following its purification to 1-O-phytyl-3-O-Galα1-6Galβ1-sn-glycerol by mass spectrometric and nuclear magnetic resonance analyses. AEGLs were ubiquitously distributed in not only green, but also red and brown marine algae; however, they were rarely detected in terrestrial plants, eukaryotic phytoplankton, or cyanobacteria.     

Genetic examination of the type specimen of Ulva australis suggests that it was introduced to Australia

‘Ana‐aosa’, one of the most common marine green algae in Japan, was described as Ulva pertusa Kjellman in 1897 from Hakodate in northern Japan. Ulva pertusa was considered to be a temperate species, with its native distributional range restricted to northeastern Asia. Although this species has been reported from various regions outside northeastern Asia, these records have been explained as non‐indigenous populations. Recently, on the basis of genetic data and nomenclatural priority, U. pertusa was synonymized with U. australis Areschoug, a species described in 1851 from specimens collected in South Australia. Based on genetic studies, Australian populations identified as U. pertusa had been considered to have originated from Japan. However, the published genetic data on U. australis in Australia have been based only on recent collections and no historical specimens have been examined. We tested the hypothesis that native (true) U. australis is an independent species of very similar morphology to U. pertusa, but that its natural domination of shoreline habitats has been suppressed by introduced populations of U. pertusa from Asia. In the present study, we extracted DNA from the type specimen of U. australis housed in the Swedish Museum of Natural History (S) and obtained DNA sequences of the chloroplast rbcL gene and the nuclear rDNA ITS2 region. Our results show that U. australis and U. pertusa are genetically virtually identical, confirming that U. pertusa is a synonym of U. australis. This suggests that the introduction of U. australis to Australia occurred by the middle of the 19th century, when the type was collected and before there was a direct shipping route between Japan and Australia. We speculate that the introduction of U. australis to Australia occurred as a secondary introduction from non‐indigenous populations in northeastern Asia, but not directly from Japan.     

α-Oxidation of Long-chain Unsaturated Fatty Acids in the Marine Green Alga Ulva pertusa

When long-chain unsaturated fatty acids such as oleic, linoleic, and linolenic acid were incubated with crude enzymes from the marine green alga Ulva pertusa, the corresponding (R)-2-hydroperoxy acids were formed with a high enantiomeric excess (>99%).     

Isolation of protoplasts from edible seaweeds

Protoplasts were isolated enzymatically from three species of Chlorophyta (Enteromorpha linza, Monostroma zostericola andUlva pertusa) with high yield and viability. An enzyme solution appropriate for protoplast isolation from the marine green algae was the following: 2% Cellulase Onozuka R-10, 1.0.M mannitol, pH 6.0. Protoplasts could not be obtained from members of Phaeophyta or Rhodophyta.     

Requisite Morphologic Interaction for Attachment between Ulva pertusa (Chlorophyta) and Symbiotic Bacteria

In order to understand the morphogenesis-inducing mechanism of Ulva pertusa by symbiotic bacteria, we observed the requisite conditions of bacteria for attachment to U. pertusa for algal morphogenesis. Non-morphogenesis-inducing bacterial mutants derived by ultraviolet irradiation did not attach onto the surface of this alga. Scanning electron microscopic observation during the process of morphogenesis in U. pertusa revealed a network-like structure formed on the algal surface within 1 week after application of bacteria. The bacteria attached onto the alga after 2 weeks of incubation. After this attachment process the morphologic change was observed in U. pertusa. Received August 7, 1997; accepted July 24, 1998.     

Nutritional interaction in an alga-barnacle association

Nitrogen is the major growth-limiting nutrient for marine algae. One potential source of nitrogen for marine algae is ammonium released by invertebrates. Many mid-intertidal reefs in northeastern New Zealand are dominated by a close association between the honeycomb barnacle Chamaesipho columna and an encusting brown alga Pseudolithoderma sp. Growth of Pseudolithoderma was enhanced in the presence of live C. columna, which released ammonium at a greater rate than the maximum rate of ammonium uptake by Pseudolithoderma. Algal tissue on barnacle tests had a lower C:N ratio than tissue located more than 2 cm from the nearest barnacle, suggesting the barnacle is an important source of nitrogen for the alga. The role of nutrient exchange in determining ecological patterns of species in marine communities is discussed.     

BACTERIA THAT INDUCE MORPHOGENESIS IN ULVA PERTUSA (CHLOROPHYTA) GROWN UNDER AXENIC CONDITIONS1

Marine foliaceous green macroalgae such as Ulva lose their typical morphology when cultured aseptically in defined synthetic media. However, after reinfection by certain marine bacteria (isolated from unialgal cultures of Ulva pertusa Kjellman), the organisms regain their typical foliaceous or tubular morphology. To investigate the morphogenesis (MG) induced in U. pertusa by bacteria, we isolated and identified bacteria with MG activity on U. pertusa and studied the distribution of such bacteria in seawater and on various marine macroalgae. We isolated 1555 bacterial strains from 18 species of marine macroalgae (six Chlorophyta, five Phaeophyta, and seven Rhodophyta), from seawater and from sediment collected at the beach at Omaezaki, Shizuoka Prefecture; Japan. Of these, 676 bacterial strains (43.5%) showed MG activity. They were classified into six bacterial groups, Flavobacterium, Vibrio, Pseudomonas, Deleya, Escherichia, and gram-positive cocci. These bacteria were ubiquitous among the samples and were not specific to U. pertusa. Several plant growth regulators had no MG activity. Filter-sterilized supernatants of culture media of MG-active bacteria strains did not induce MG. Cocultivation of Ulva with active bacterial strains is so far the only way to induce the MG effect, which suggests that for MG direct contact between Ulva and the bacterial strain is necessary.     

Photosynthesis and photoinhibition of two green macroalgae with contrasting habitats

We evaluated light-related traits ofUlva pertusa Kjellman from the intertidal and upper subtidal zones, and ofUmbraulva japonica (Holmes) Bae & Lee (formerlyUlva japonica) within its upper growth limit of 10 m, in Korean coastal regions.U. pertusa showed significantly higher maximum photosynthetic rates, photosynthetic efficiency, saturating irradiances, and total pigment contents. However, green light-use efficiency at limiting irradiances was notably higher inU. japonica, possibly due to the presence of green light absorbing pigments like siphonaxanthin and siphonein. Non-enzymatic antioxidation capacity as determined by DPPH (α,α-diphenyl-β-picrylhydrazyl) radical scavenging rate was markedly higher inU. pertusa (50.49%) than inU. japonica (8.85%). Both species showed a substantial decrease in optimal photosystem (PS) II efficiency (F_v/ F_m) with increasing PAR doses despite the degree of photoinhibition being more marked inU. japonica. After 24 h in dim light,U. pertusa rapidly and almost fully recovered F_v/ F_m within an hour whileU. japonica exhibited slow and incomplete PSII recovery over the full recovery period. A significant depression in photosynthetic activity with monochromatic UV-B radiation was observed inU. japonica only, followed by slight recovery in dim light. Light use efficiency and high irradiance tolerance may be important ecological axes along which the niche separation of green foliose algae occurs in Korean coastal waters.     

Carbon budgets for a phytoplanktivorous fish fed three different unialgal populations

Summary The filter feeding blue tilapia, Tilapia aurea, was fed three different algae. Blue tilapia ingestion of two green algae, Chlamydomonas sp. and Ankistrodesmus falcatus and the filamentous blue-green alga, Anabaena flos-aquae, ranged from 21%–89% of the available cells. There were significant differences in the assimilation of algal carbon by the fish depending on the alga fed; A. flos-aquae was the easiest to assimilate (83%). The fish respired significantly less of the Chlamydomonas sp. ingested carbon (15%). The gross growth efficiency of fishes fed either green alga was not significantly different (22%–24%), but these efficiencies were significantly less than the gross growth efficiency of fish fed A. flos-aquae (46%). The carbon budgets for fish feeding on the green algae were similar to that constructed from the literature for a congener fed a mixed algae diet. However, the assimilation component of the budget for blue tilapia fed A. flos-aquae was 2 times greater than that of the literature budget.     

Distribution of an enzyme system producing seaweed flavor: conversion of fatty acids to long-chain aldehydes in seaweeds

The long chain aldehyde-forming enzyme (LCAE) activity that catalyzes formation of long chain aldehydes, such as (8Z, 11Z, 14Z)-heptadecatrienal, (8Z, 11Z)-heptadecadienal, (8Z)-heptadermal, (7Z, 10Z, 13Z)-hexadecatrienal and pentadecanal from linolenic acid, linoleic acid, oleic acid and palmitic acid, in that order, occurs in a wide range of green, brown and red seaweeds. The LCAE activity increased with maturation of juvenile fronds of Enteromorpha sp. in culture. Thus, cultivation of seaweeds for flavor foods is of interest. The release of long chain aldehydes from the thallus into the medium was confirmed by a quantitative high performance liquid chromatography of volatile compounds, using a closed loop stripping technique, during the culture of the green alga, Ulva pertusa. This finding suggests physiological roles of long chain aldehydes and LCAE activity in marine ecosystems.     

Effect of bacterial biofilms formed on fouling-release coatings from natural seawater and Cobetia marina,on the adhesion of two marine algae

Previous studies have shown that bacterial biofilms formed from natural seawater (NSW) enhance the settlement of spores of the green alga Ulva linza, while single-species biofilms may enhance or reduce settlement, or have no effect at all. However, the effect of biofilms on the adhesion strength of algae, and how that may be influenced by coating/surface properties, is not known. In this study, the effect of biofilms formed from natural seawater and the marine bacterium Cobetia marina, on the settlement and the adhesion strength of spores and sporelings of the macroalga U. linza and the diatom Navicula incerta, was evaluated on Intersleek® 700, Intersleek® 900, poly(dimethylsiloxane) and glass. The settlement and adhesion strength of these algae were strongly influenced by biofilms and their nature. Biofilms formed from NSW enhanced the settlement (attachment) of both algae on all the surfaces while the effect of biofilms formed from C. marina varied with the coating type. The adhesion strength of spores and sporelings of U. linza and diatoms was reduced on all the surfaces biofilmed with C. marina, while adhesion strength on biofilms formed from NSW was dependent on the alga (and on its stage of development in the case of U. linza), and coating type. The results illustrate the complexity of the relationships between fouling algae and bacterial biofilms and suggest the need for caution to avoid over-generalisation.     

Characterization of Chlorophyll–protein Complexes Isolated from Two Marine Green Algae, Bryopsis maxima and Ulva pertusa, Growing in the Intertidal Zone

Three Chl–protein complexes were isolated from thylakoid membranes of Bryopsis maxima and Ulva pertusa, marine green algae that inhabit the intertidal zone of the Pacific Ocean off the eastern coast of Japan by dodecyl-β-d-maltoside polyacrylamide gel electrophoresis. The slowest-moving fractions showed low Chl a/b and Chl/P-700 ratios, indicating that this fraction corresponds to complexes in PS I, which is large in both algae. The intermediate and fastest-moving fractions showed the traits of PS II complexes, with some associated Chl a/b–protein complexes and LHC II, respectively. The spectral properties of the separated Chl–proteins were also determined. The absorption spectra showed a shallow shoulder at 540 nm derived from siphonaxanthin in Bryopsis maxima, but not in Ulva pertusa. The 77 K emission spectra showed a single peak in Bryopsis maxima and two peaks in Ulva pertusa. Besides the excitation spectra indicated that the excitation energy transfer to the PS I complexes differed quite a lot higher plants. This suggested that the mechanisms of energy transfer in both of these algae differ from those of higher plants. Considering the light environment of this coastal area, the large size of the antennae of PS I complexes implies that the antennae are arranged so as to balance light absorption between the two photosystems. In addition, we discuss the relationships among the photosystem stoichiometry, the energy transfer, and the distribution between the two photosystems.     

Investigations on the feeding habits of the rocky-shore miteHyadesia fusca (Acari: Astigmata: Hyadesiidae): diet range, food preference, food quality, and the implications for distribution patterns

Within the food web of estuarine and marine rocky shore ecosystems phytophagous mites of terrestrial and marine origin constitute an important part as grazers on algae and as a food source for certain arthropods, especially zoophagous mites. This investigation deals with the feeding biology ofHyadesia fusca taking as an example a population located on an artificial rocky shore of the middle Weser estuary in Northern Germany. The species is characterized by a broad diet range; in feeding experiments diatoms, lichens, detritus as well as blue, red and green algae were accepted. Even analyses of faecal pellets produced by field specimen suggest a non-specific feeding habit. However, the influence of certain diets on mortality, offspring number and rearing success showed that the food quality differs significantly. The most suitable food, the UlvaceaeBlidingia, was clearly preferred in a series of pairwise choice tests. These findings correlate with the vertical zonation of the field population i.e.: higher population densities in the vegetation zone dominated byBlidingia. It can be concluded that in addition to abiotic factors food supply could play an important role for distribution patterns of phytophagous mites.     

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 thealgae, thereby demonstrating the release of the non-polar metabolites involved in antifouling processes. Thedifferences 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 μm² 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.     

Green urchin as a significant source of fecal particulate organic matter within nearshore benthic ecosystems

The role of green sea urchin Strongylocentrotus droebachiensis as a source of fecal particulate organic matter (POM) for the benthic nearshore ecosystems has been studied over a 3.5-month period. Three macroalgae were tested as food sources: Alaria esculenta, Laminaria longicruris and Ulvaria obscura. Urchins were fed ad libitum with either a single alga species or a mixture of all three algae. Consumption and defecation rates were determined as well as the feces/alga ratio in term of biomass and biochemical composition. Consumption rate increased exponentially with urchin size and also varied with alga species. In the single alga trial, consumption rate was higher for both brown algae (Laminaria and Alaria) compared to Ulvaria. Urchins feeding on the mixture of algae maintained their total ingestion rate (sum of the three algae) at the same level to those feeding on a single alga diet. The mixed algae trial showed that urchins clearly preferred Laminaria (72% of total ingestion) over Alaria (22%) and Ulvaria (6%). The defecation rate was tightly correlated with the food consumption rate and thus increased with urchin size. On average, 75% of the ingested algal biomass was released as fecal POM. The percentage of food defecated changed with alga species, with the highest value for Alaria (81%) and the lowest for Laminaria (67%). The percentage of food defecated by urchins feeding on the mixture of algae was generally comparable to those feeding on single alga diet. Biochemical composition (in soluble carbohydrates, proteins and lipids) of urchin fecal POM reflected that of the algae content. From 40% to 80% of macronutrients in algal food persisted in fecal matter. This proportion varied with the alga species and macronutrient considered. This study shows that the green sea urchin plays a significant role in the production of POM within nearshore benthic ecosystems, and it is a potentially nutritious food source for detritivores.     

Photosynthesis and growth of Ulva ohnoi and Ulva pertusa (Ulvophyceae) under high light and high temperature conditions,and implications for green tide in Japan

The macroalga Ulva ohnoi constitutes a considerable fraction of green tides in coastal areas of Japan, but little is known about the physiological characteristics of this species. To investigate the environmental factors that promote the formation of green tides, we tested the responses of U. ohnoi and another common Japanese species, Ulva pertusa, to various levels of irradiance at different water temperatures. Because the two species are morphologically similar, we identified them using the PCR‐restriction fragment length polymorphism method. Under laboratory conditions, we evaluated the photosynthetic, dark respiration, and relative growth rate at a range of water temperatures (5 to 35°C) and photosynthetically active radiation (0 to 1000 μmol photons m^?2 s^?1). The maximum gross photosynthetic rate of U. ohnoi was larger than that of U. pertusa. The dark respiration rates revealed no significant differences among the species and temperature conditions. At 500 μmol photons m^?2 s^?1, the relative growth rate of U. ohnoi was larger than that of U. pertusa in higher temperature and the difference was the largest at 20°C. The estimated compensation irradiance and estimated saturation irradiance of U. ohnoi and U. pertusa ranged from 0.709 to 5.510 and 40.530 to 58.674 μmol photons m^?2 s^?1, which were lower than those in other intertidal green macroalgae, from 6 to 11 and 50 to 82 μmol photons m^?2 s^?1, respectively. Thus, U. ohnoi which exists as free‐floating near the water surface and accumulating inside the green tide can survive extensively in the water column of the intertidal zone, furthermore, the species can maintain rapid growth in this situation. Therefore, as a result of this study, it is suggested that the ecological success of U. ohnoi in shallow waters such as the tidal flats, estuarine, and coasts of the inner bay in comparison with U. pertusa.     

A multi-locus time-calibrated phylogeny of the siphonous green algae

The siphonous green algae are an assemblage of seaweeds that consist of a single giant cell. They comprise two sister orders, the Bryopsidales and Dasycladales. We infer the phylogenetic relationships among the siphonous green algae based on a five-locus data matrix and analyze temporal aspects of their diversification using relaxed molecular clock methods calibrated with the fossil record. The multi-locus approach resolves much of the previous phylogenetic uncertainty, but the radiation of families belonging to the core Halimedineae remains unresolved. In the Bryopsidales, three main clades were inferred, two of which correspond to previously described suborders (Bryopsidineae and Halimedineae) and a third lineage that contains only the limestone-boring genus Ostreobium. Relaxed molecular clock models indicate a Neoproterozoic origin of the siphonous green algae and a Paleozoic diversification of the orders into their families. The inferred node ages are used to resolve conflicting hypotheses about species ages in the tropical marine alga Halimeda.     

The green alga Dicytosphaeria ocellata and its organic extracts alter natural bacterial biofilm communities

Surfaces immersed in the marine environment are under intense fouling pressure by a number of invertebrates and algae. The regulation of this fouling can often be attributed to the bacterial biofilm that quickly develops on the surface of any available substratum in the ocean. The bacterial community composition on the surface of the green alga Dictyosphaeria ocellata was investigated and compared to those found on two other green algae, Batophora oerstedii and Cladophoropsis macromeres, and on a reference surface from three sites along the Florida Keys. Although the bacterial community composition of D. ocellata was not consistent across the sites, it was significantly different from the other algae and the reference surface at two of the three sites tested. Methanol extracts of D. ocellata significantly affected the abundance of bacteria and composition of the bacterial community on Phytagel? plates when compared to solvent controls, suggesting that the alga regulates the bacterial community by producing active metabolites.