Periodic Studies of the Proteins, Peptides, and Free Amino-Acids in Enteromorpha prolifera f. capillaris and Ulva lactuca var. rigida

The technique of circular paper chromatography was used in theanalysis of free amino-acids and amino-acid constituents ofproteins and peptides in Enteromorpha prolifera f. capillarisand Ulva lactuca var. rigida collected from Bombay at fortnightlyintervals. The amino-acids present in the proteins at different stagesof algal grwoth remained the same; but quantitative variationsin composition were usually observed, and these were independentof the age of the plant. Significant quantitative and qualitative variations were observedin the amino-acids of the peptides and in the free state, butvariations were usually independent of the age of the plant.However, in Enteromorpha prolifera f. capillaris, leucine(s),proline, and valine in the peptides, and serine in the freestate were found to increase with the age of the plant. Of the amino-acids recorded in the proteins, homocystine wasnot found in the peptide hydrolysates or among the free amino-acids.Tryptophan was absent from the peptides. Cysteric acid was foundin the peptides and in the free state, and asparagine in thefree state only.     

A new heteronemertean from the Argentine coast of the Southern Atlantic

A new heteronemertean is described, Gastropionsanmatiensis n. gen. et n. sp. from Caleta de LosLoros, San Matías Gulf, Argentine Republic(41& deg;S; 64° 05 W). G. sanmatiensis is mainly defined by: (1) the structure ofits proboscis sheath; (2) enteronephridia and exonephridiasituated in the anterior part of the oesophagus; (3) a sphincterbetween the oesophagus and the intestine; (4) anintestinal sphincter. This lineid heteronemerteanseems to be very well adapted to oesophagealrespiration and excretion.     

Comparison of different extractive procedures for proteins from the edible seaweeds Ulva rigida and Ulva rotundata

Proteins have been extracted from the edible seaweeds Ulva rigida Agardh and Ulva rotundata Bliding using classical or enzymatic procedures. The protocols using NaOH under reductive conditions or a two-phase system (PEG/K₂CO₃) produced the best protein yields. The cleavage or the limitation of the linkages between proteins and polysaccharides caused by these experimental conditions probably explains the efficiency of these protocols. In SDS PAGE, the protein fraction obtained after NaOH extraction from U. rotundata is characterised by the presence of three major bands with apparent molecular weights of 45 600, 31 800 and 18 600. The protein fraction from U. rigida presents two specific bands with apparent molecular weights of about 27 000 and 12 000. These fractions are mainly rich in aspartic and glutamic acids, alanine, glycine and contain few hydroxyproline residues (0.91–2.44% total amino acid content). The use of cellulase does not significantly improve the extraction of algal proteins in comparison with the blank procedure (without enzymes). The weak accessibility of the substrates in the intact cell wall could explain these experimental data. The improvement of protein yield after the use of the polysaccharidase mixture (-glucanase, hemicellulase, cellulase) partially confirms this hypothesis.     

Identification by SDS PAGE of green seaweeds ( Ulva and Enteromorpha) used in the food industry

SDS PAGE was tested as an analytical tool for the identification of fourgreen algae (Ulva rigida, U. rotundata, Enteromorphaintestinalis, E. compressa) used as food ingredients. A referencepattern composed of the bands present all year long was performed foreach species. The pattern for Ulva rotundata consists of 7 bandslocated between 69.9 and 15.5 kDa with the presence of triplicate bandsat 29.5, 26.3 and 22.9 kDa. The pattern for Ulva rigida is consistsof three bands with apparent molecular weights of 68.5, 56.4 and 44.7kDa. The Enteromorpha compressa pattern is characterised by sixbands located between 65.8 and 19.8 kDa. A double band withmolecular weights of 23.1 and 23.9 distinguished this pattern from theothers. Six bands situated between 66.4 and 19.4 kDa with a specifictriplicate band of 25.9, 23.9 and 22.5 kDa constituted the specific patternof Enteromorpha intestinalis. SDS PAGE appears to be suitable forthe identification of green seaweed foods.     

Protein content, amino acid composition and nitrogen-to-protein conversion factors of Ulva rigida and Ulva capensis from natural populations and Ulva lactuca from an aquaculture system, in South Africa

The South African abalone aquaculture industry is expanding and there is a lack of information on the nutritional profiles of the seaweeds that are used as feed. The current study quantified the protein contents of Ulva rigida and Ulva capensis from natural populations and Ulva lactuca from a commercial, integrated seaweed/abalone aquaculture system. Three methods of protein quantification were used: the Bradford method, crude protein, and specific nitrogen to protein (N-Prot) conversion factors, and results were compared to each other. The results showed that values obtained with the “traditional” conversion factor of 6.25 were on average higher than Bradford values by factors of 64.1 % in U. capensis, 77.1 % in U. rigida and 58.9 % in U. lactuca. This pattern is in line with other published work on seaweed and microalgae. Analyses of amino acid composition showed that aspartic acid was the most abundant amino acid and that these species were also rich in glycine and alanine but poor in histidine, methionine and cysteine. N-Prot factors were as follows: U. capensis, 5.58; U. rigida, 5.12 and U. lactuca, 5.65. An average N-Prot factor of 5.45 provides a more accurate estimate of the protein content of the Ulva species studied than the “traditional” factor of N?×?6.25.     

Antifouling potential of lubricious, micro-engineered, PDMS elastomers against zoospores of the green fouling alga Ulva (Enteromorpha)

The settlement and release of Ulva spores from chemically modified, micro-engineered surface topographies have been investigated using poly(dimethyl siloxane) elastomers (PDMSe) with varying additions of non-network forming poly(dimethyl siloxane) based oils. The topographic features were based on 5 microns wide pillars or ridges separated by 5, 10, or 20 microns wide channels. Pattern depths were 5 or 1.5 microns. Swimming spores showed no marked difference in settlement on smooth surfaces covered with excess PDMS oils. However, incorporation of oils significantly reduced settlement density on many of the surfaces with topographic features, in particular, the 5 microns wide and deep channels. Previous results, confirmed here, demonstrate preferences by the spores to settle in channels and against pillars with spatial dimensions of 5 microns, 10 microns and 20 microns. The combination of lubricity and pillars significantly reduced the number of attached spores compared to the control, smooth, unmodified PDMSe surfaces when exposed to turbulent flow in a flow channel. The results are discussed in relation to the energy needs for spores to adhere to various surface features and the concepts of ultrahydrophobic surfaces. A factorial, multi-level experimental design was analyzed and a 2nd order polynomial model was regressed for statistically significant effects and interactions to determine the magnitude and direction of influence on the spore density measurements between factor levels.     

Red-light and blue-light photoreceptors controlling chlorophyll a synthesis in the red alga Porphyra umbilicalis and in the green alga Ulva rigida

Chlorophyll synthesis is stimulated by red light in the green alga Ulva rigida C. Ag. and in the red alga Porphyra umbilicalis (L.) Kützing. Because the effect of red light showed some far-red reversibility in successive red and far-red light treatments, the involvement of phytochrome or a phytochrome-like photoreceptor is suggested. The extent of the response is dependent on exposure and photon fluence rate of red-light pulses. In addition to the effect of red light, a strong stimulation of chlorophyll synthesis by blue light was only observed in Ulva rigida. The effect of blue light shows also some far-red reversibility. In the green alga the accumulated chlorophyll is higher after blue light pulses than after red light pulses. In Porphyra umbilicalis , however, the contrary is observed. In Ulva rigida the involvement of a blue light photoreceptor in addition to phytochrome or a phytochrome-like photoreceptor is proposed. The different responses to red and blue light in both algae are explained in terms of their adaptation to the natural light environment.     

Ulva rigida in the future ocean: potential for carbon capture,bioremediation and biomethane production

Ulva species have been considered as ideal candidates for carbon capture, bioremediation and biofuel production. However, little is known regarding the effects of simultaneous ocean warming, acidification and eutrophication on these capacities. In this study, Ulva rigida was cultivated under two levels of: temperature (14 °C (LT) and 18 °C (HT)); pH (8.10 and 7.70) by controlling pCO₂ (LC, HC respectively); and nutrients (low (LN) – 50 μm N and 2.5 μm P and high (HN) – 1000 μm N and 50 μm P) for 6 weeks. During the first week of cultivation, HT, HC and HN increased biomass by 38.1%, 17.1% and 20.8%, respectively, while the higher temperature led to negative growth in weeks 2, 4 and 6 due to reproductive events. By the end of the cultivation, biomass under HTHCHN was 130.4% higher than the control (LTLCLN), contributing to a higher carbon capture capacity. Although the thalli at HT released nutrients to seawater in weeks 2, 4 and 6, the HTHCHN treatment increased the overall nitrate uptake rate over the cultivation period by 489.0%. The HTHCHN treatment also had an increased biochemical methane potential and methane yield (47.3% and 254.6%, respectively). Our findings demonstrate that the capacities for carbon and nutrient capture, and biomethane production of U. rigida in the future ocean may be enhanced, providing important insight into the interactions between global change and seaweeds.     

Molecular phylogenetic analyses of the Japanese Ulva and Enteromorpha (Ulvales, Ulvophyceae), with special reference to the free-floating Ulva

In order to elucidate the species composition of free‐floating Ulva that cause green tide in several bays in Japan, and to clarify the generic status of Ulva and Enteromorpha (Ulvales, Ulvophyceae), the nuclear encoded internal transcribed spacer (ITS) region including the 5.8S gene and the plastid encoded large subunit of ribulose‐1, 5‐bisphosphate carboxylase/ oxgenase (rbcL) gene sequences for 15 species were determined. Both ITS and rbcL analyses indicate that free‐floating Ulva samples are divided into four different lineages that correspond to Ulva lactuca Linnaeus, U. pertusa Kjellman, U. armoricana Dion etal. and U. fasciata Delile. These four species are distinguished by cell morphology including the arrangement of cells, the shape and size of cells and the position of chloroplasts. Molecular data also indicated that Ulva and Enteromorpha are not separated as respective monophyletic groups within a large monophyletic clade and congeneric as shown by previous molecular studies using the ITS sequences alone. This strongly suggests that these genera are congeneric and Enteromorpha should be reduced to the synonym of Ulva.     

Purification, characterization, and cDNA cloning of a novel lectin from the green alga, Codium barbatum

A novel lectin (CBA) was isolated from the green alga, Codium barbatum, by conventional chromatographic methods. The hemagglutination-inhibition profile with sugars and glycoproteins indicated that CBA had preferential affinity for complex type N-glycans but not for monosaccharides, unlike the other known Codium lectins specific for N-acetylgalactosamine. CBA consisted of an SS-linked homodimer of a 9257-Da polypeptide containing seven cysteine residues, all of which were involved in disulfide linkages. The cDNA of the CBA subunit coded a polypeptide (105 amino acids) including the signal peptide of 17 residues. The calculated molecular mass from the deduced sequence was 9705 Da, implying that the four C-terminal amino acids of the CBA proprotein subunit were post-translationally truncated to afford the mature subunit (84 amino acids). No significantly similar sequences were found during an in silico search, indicating CBA to be a novel protein. CBA is the first Codium lectin whose primary structure has been elucidated.     

Roughness-dependent removal of settled spores of the green alga Ulva (syn. Enteromorpha) exposed to hydrodynamic forces from a water jet

Topographic features change the hydrodynamic regime over surfaces subjected to flow. Hydrodynamic microenvironments around topographic structures may have consequences for recruitment and removal of propagules of marine benthic organisms. The settlement and adhesion of zoospores from the green alga Ulva linza (syn. Enteromorpha linza) to defined topographies was investigated. A range of topographic size scales (Rz: 25-100 microm) was manufactured from plankton nets, creating patterns with ridges and depressions. The topographic scales span a roughness similar to that of natural substrata and antifouling coatings. Spores were removed from the surfaces by a calibrated water jet. Fewer spores were removed from the smallest topographic structure tested (Rz: 25 microm) compared to both the smooth (Rz: 1) and the roughest (Rz: 100 microm) structures. Zoospores that settled in depressions were less likely to be removed compared to spores on the ridges. The results in terms of the interaction between surface topography and hydrodynamic forces have implications for both natural substrata exposed to wave action and antifouling surfaces on ships' hulls. The possible effects of topography on increasing zoospore adhesion and offering a refuge from hydrodynamic forces are discussed.     

Seed dispersal and population structure in Vriesea gigantea,a bromeliad from the Brazilian Atlantic Rainforest

Seed dispersal, population structure and the mating system of plant species can have great consequences on the genetic structure of populations. Vriesea gigantea is a bromeliad from southeastern Brazil which is self‐compatible and pollinator dependent for fruit set. Its populations are fertile in terms of the production of flowers, pollen, fruits and seeds. To assess the importance of seed supply for gene flow, colonization and distribution of adult individuals, the seed dispersal and population structure of V. gigantea were studied. Seeds are dispersed over short distances; most seeds land close to the mother plant. This pattern coincides with the reported aggregate distribution of bromeliad seedlings. Population structure results showed high seedling recruitment, because 51.3% developed into adults, although few juveniles reached this stage. This result is different from that for other bromeliad species from different habitat conditions. Seed dispersal and population structural patterns are consistent with previous molecular studies, revealing that V. gigantea populations are genetically structured, with low gene flow and a moderate outcrossing rate. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164 , 317–325.     

Molecular phylogenetic evidence for a reversible morphogenetic switch controlling the gross morphology of two common genera of green seaweeds, Ulva and Enteromorpha.

Ulva and Enteromorpha are two of the most common, ubiquitous, and environmentally important genera of green seaweeds. They are widely regarded as easily distinguishable because of their dramatically different morphologies: Ulva species are flat, lettucelike blades two cell layers thick, and Enteromorpha species form hollow liquid- or gas-filled tubes one cell thick, which may also be highly branched. We present molecular phylogenetic analyses of nuclear ribosomal RNA ITS sequences from 39 samples representing 21 purported species within these two genera. The results clearly indicate that the two genera are not respectively monophyletic and that the characteristic Ulva and Enteromorpha morphologies have arisen independently several times throughout the evolutionary diversification of the group. The analyses demonstrate that this radical change in gross morphology can also happen within clades exhibiting sequence divergence typical of conspecific assemblages of this group. We suggest that this morphological flexibility is the result of some form of developmental switch that results in either blades or tubes, but that this putative switch must be activated relatively infrequently, since there is evidence that some lineages have retained their form for significant periods. This discovery suggests a possible new model system for study of the molecular mechanisms involved in the interplay between environmental stimuli and plant development.     

Abundance and distribution of Ulva microscopic propagules associated with a green tide in the southern coast of the Yellow Sea

The presence of Ulva microscopic propagules may play an important role in the rapid development of high-biomass blooms of green algae in the Yellow Sea. Six cruises were conducted, to determine the abundance and distribution of Ulva microscopic propagules associated with a green tide that developed in the southern coastal waters of the Yellow Sea from April to August, 2012. Results indicated that Ulva microscopic propagules were widespread in these waters, with the highest density being up to 4800 ind. L⁻¹, prior to the appearance of the green tide in April. High densities were also widely distributed along the coast during May and June, after the appearance of the floating green tide. The quantity of Ulva microscopic propagules significantly decreased when the floating green tide declined in July, reaching densities of up to 162 ind. L⁻¹, following the disappearance of the floating green tide in August. Quantitative studies on the distribution patterns of Ulva microscopic propagules along the southern coast of the Yellow Sea indicated a significant correlation between density and salinity, turbidity and nutrient concentrations. Temporal and geographical distribution patterns of Ulva microscopic propagules were also significantly affected by the presence of a large biomass of attached, or floating, Ulva species algae.     

Use of the green alga Ulva rigida C. Agardh as an indicator species to reassess metal pollution in the Thermaikos Gulf, Greece, after 13 years

The concentrations of metals (Mn, Pb, Fe, Zn, Cu, Cd,Co, Ni, Cr, Na, K, Ca, Mg) were determined in thegreen alga Ulva rigida, in the sediment andseawater of Thermaikos Gulf (Greece) during monthlysamplings in 1994–1995. This Gulf is the recipientof domestic and industrial effluents. Pb, Fe, Cu, Coand Cr concentrations in U. rigida at the studyarea were higher than those 13 years earlier andapparently came from different sources than those forZn, Cd and Ni. The relative abundance of metals inthe alga decreased in the order: Mg > Na > K >Ca > Pb > Fe > Mn > Zn > Cr, Cu > Ni >Co > Cd. Only Cu concentrations in the alga fromKalochori and Cd ones from Viamyl showed significantseasonal changes. Cu and Cd concentrations ingeneral followed the same pattern of variation, withminimum values in winter-spring. This pattern isdiscussed in relation to growth dynamics and tissueage. Only Pb concentrations in the alga showed asignificant positive correlation with concentrationsin the seawater. There were both positive andnegative correlations among some metals in the alga. It is concluded that U. rigida can be used as anindicator species, especially for Pb.     

Interaction of Photoprotective and Acclimation Mechanisms in Ulva rigida (Chlorophyta) in Response to Diurnal Changes in Solar Radiation in Southern Chile

Species of the genus Ulva (Chlorophyta) are regarded as opportunistic organisms, which efficiently adjust their metabolism to the prevailing environmental conditions. In this study, changes in chlorophyll‐a fluorescence‐based photoinhibition of photosynthesis, electron transport rates, photosynthetic pigments, lipid peroxidation, total phenolic compounds, and antioxidant metabolism were investigated during a diurnal cycle of natural solar radiation in summer (for 12 h) under two treatments: photosynthetically active radiation (PAR: 400–700 nm) and PAR+ ultraviolet (UV) radiation (280–700 nm). In the presence of PAR alone, Ulva rigida showed dynamic photoinhibition, and photosynthetic parameters and pigment concentrations decreased with the intensification of the radiation. On the other hand, under PAR+UV conditions a substantial decline up to 43% was detected and an incomplete fluorescence recovery, also, P‐I curve values remained low in relation to the initial condition. The phenolic compounds increased their concentration only in UV radiation treatments without showing a correlation with the antioxidant activity. The enzimatic activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX) increased over 2‐fold respect at initial values during the onset of light intensity. In contrast, catalase (CAT) increased its activity rapidly in response to the radiation stress to reach maxima at 10 a.m. and decreasing during solar. The present study suggests that U. rigida is capable of acclimating to natural radiation stress relies on a concerted action of various physiological mechanisms that act at different times of the day and under different levels of environmental stress.     

A simple method for mass isolation of protoplasts from species of Monostroma, Enteromorpha and Ulva (Chlorophyta, Ulvales)

A simple enzyme mixture containing 2% Cellulase Onozuka R–10 and1% Macerozyme R–10 prepared in deionised water supplemented with 3% NaCland 1 mM CaCl₂ was developed for isolating rapidlyprotoplasts from different species of Monostroma,Enteromorpha and Ulva. The yield fordifferent species of Monostroma ranged from 9.6 ×10⁶ to 10.2 × 10⁶ cells g^–1f. wt thallus, and forEnteromorpha from 3.48 × 10⁶ to 11.7× 10⁶ cells g^–1 f. wt and forUlva from 4.58 × 10⁶ to 26.8 ×10⁶ cells g^–1 f. wt. The overallregeneration rate of the protoplasts isolated was usually > 90% and showednormal morphogenesis. The method yields rapid mass production of viableprotoplasts with high regeneration rates.     

Outplanting time and methodologies related to mariculture of the edible kelp Undaria pinnatifida in the Atlantic coast of Spain

Little is known about offshore cultivation of the edible seaweed Undaria pinnatifida outside Asian waters. However, currently its commercial cultivation is being considered in the North Western Atlantic coast of Spain (Galicia). The effect of outplanting time and methodologies related to the cultivation of U. pinnatifida in this area was studied. Biomass yield, morphological features, and growth rate were examined on sporophytes outplanted from October to December at the same site in a coastal bay. Experiments were conducted over two years, applying different harvesting and cultivation methods. Results indicate that the most suitable months for outplanting were October and November, with larger biomass production as well as improved sporophyte quality (i.e., morphology). Cultivations from October to December ended when most of the sporophytes had developed sporophylls (reproductive structures) and this occurred between early and late April in all the outplanting trials. The similar harvest time for all plants suggests that harvesting time is not related to outplanting period but should be mainly influenced by the photoperiod. These results suggest a single harvest as the most appropriate method. Moreover, results also showed that cultivation was successful using the two tested methods: seedling string all along the culture rope and seedling strings inserted at regular intervals into the rope. Finally, this paper also discusses the suitability of two different systems of offshore farming: in long-line and in vertical rope.     

Chemical and in situ characterization of macromolecular components of the cell walls from the green seaweed Codium fragile

A comprehensive analysis of the carbohydrate-containing macromolecules from the coencocytic green seaweed Codium fragile and their arrangement in the cell wall was carried out. Cell walls in this seaweed are highly complex structures composed of 31% (w/w) of linear (1-->4)-beta-D-mannans, 9% (w/w) of pyruvylated arabinogalactan sulfates (pAGS), and low amounts of hydroxyproline rich-glycoprotein epitopes (HRGP). In situ chemical imaging by synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy and by immunolabeling using antibodies against specific cell wall carbohydrate epitopes revealed that beta-d-mannans and pAGS are placed in the middle part of the cell wall, whereas HRGP epitopes (arabinogalactan proteins (AGPs) and extensins) are located on the wall boundaries, especially in the utricle apical zone. pAGS are sulfated at C-2 and/or C-4 of the 3-linked beta-L-arabinopyranose units and at C-4 and/or C-6 of the 3-linked beta-D-galactopyranose residues. In addition, high levels of ketals of pyruvic acid were found mainly at 3,4- of some terminal beta-D-Galp units forming a five-membered ring. Ramification was found at some C-6 of the 3-linked beta-D-Galp units. In agreement with the immunolabeled AGP epitopes, a nonsulfated branched furanosidic arabinan with 5-linked alpha-L-Araf, 3,5-linked alpha-L-Araf, and terminal alpha-L-Araf units and a nonsulfated galactan structure composed of 3-(3,6)-linked beta-D-Galp residues, both typical of type-II AG glycans were found, suggesting that AGP structures are present at low levels in the cell walls of this seaweed. Based on this study, it is starting to emerge that Codium has developed unique cell wall architecture, when compared, not only with that of vascular plants, but also with other related green seaweeds and algae.     

Some properties and amino acid sequence of plastocyanin from a green alga, Ulva arasakii

Plastocyanin was purified from a multicellular, marine green alga, Ulva arasakii, by conventional methods to homogeneity. The oxidized plastocyanin showed absorption maxima at 252, 276.8, 460, 595.3, and 775 nm, and shoulders at 259, 265, 269, and 282.5 nm; the ratio A276.8/A595.3 was 1.5. The midpoint redox potential was determined to be 0.356 V at pH 7.0 with a ferri- and ferrocyanide system. The molecular weight was estimated to be 10,200 and 11,000 by SDS-PAGE and by gel filtration, respectively. U. arasakii also has a small amount of cytochrome c6, like Enteromorpha prolifera. The amino acid sequence of U. arasakii plastocyanin was determined by Edman degradation and by carboxypeptidase digestion of the plastocyanin, six tryptic peptides, and five staphylococcal protease peptides. The plastocyanin contained 98 amino acid residues, giving a molecular weight of 10,236 including one copper atom. The complete sequence is as follows: AQIVKLGGDDGALAFVPSKISVAAGEAIEFVNNAGFPHNIVFDEDAVPAGVDADAISYDDYLNSKGETV VRKLSTPGVY G VYCEPHAGAGMKMTITVQ. The sequence of U. arasakii plastocyanin is closet to that of the E. prolifera protein (85% homology). A phylogenetic tree of five algal and two higher plant plastocyanins was constructed by comparing the amino acid differences. The branching order is considered to be as follows: a blue-green alga, unicellular green algae, multicellular green algae, and higher plants.