Ecological studies on Ulva lactuca L. from Veraval,India

Ulva lactuca L., at Veraval (20° 54N and 70° 22E) on the western coast of India, grows in the intertidal belt from June to late February. During the summer months of March, April and May, the species dries up, leaving rhizoidal fragments for perennation. High values of density, phytomass, frequency, weight, and volume have been recorded during September and January. Accumulation of phytomass appears to be chiefly density dependent. High dissolved oxygen content and low temperatures of the surface sea water favoured enhanced growth and, consequently, more phytomass.     

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.     

Ulva lactuca as bioindicator of metal contamination in intertidal waters in Hong Kong

The use of Ulva lactuca L. as an indicator of metal contamination was assessed by analysing the levels of Mn, Fe, Ni, Cu, Zn, Cd and Pb in the alga collected from 24 intertidal sites around the Island of Hong Kong. Twelve of the sites are in the rural southern parts of the Island where the coastal waters are relatively clean. The remaining 12 sites are located in the north and within Victoria Harbour which receives, apart from industrial effluents, untreated domestic sewage from a population of some 3.5 million. The mean levels of Mn, Fe, Ni, Cu, Zn and Pb in Ulva from the urban sites were respectively 4.0, 4.6, 1.8, 2.3, 2.4 and 4.6 folds those from the rural sites. However, similar levels of Cd were found in the alga amongst all the sites. Locations of high levels of metal contamination, particularly to the eastern end of the Harbour, have been identified. Preliminary results indicate that Ulva is a good indicator of Mn, Fe, Cu, Zn and Pb contaminations.     

Mineral element content in Ulva lactuca L. with reference to eutrophication in Hong Kong coastal waters

The amounts of tissue nitrogen, phosphorus, potassium, sodium, calcium and iron were estimated in the green alga Ulva lactuca L. collected from 9 rural and 14 urban littoral sites in the waters around Hong Kong Island during 1978 and 1979. The mean levels of tissue nitrogen and phosphorus were respectively 65% and 87% more in urban sites than in rural ones. Very significant correlation (r = 0.920) was found between the logarithmic concentration of seawater inorganic nitrogen and that of tissue nitrogen. The same applied to soluble reactive phosphorus in seawater and tissue phosphorus (r = 0.886). The levels of potassium, sodium and calcium in the alga were relatively uniform. However considerable variation in the level of iron was detected. The potential use of Ulva as an indicator species for eutrophication is discussed.     

Perturbation of monovalent cation composition in Ulva lactuca by cadmium, copper and zinc

Discs of thallus cut from the macroalga Ulva lactuca were incubated in filtered seawater containing cadmium, zinc, copper or cobalt (30 m). The metal uptake rates differed for each metal in the order Cu > Zn > Cd > Co. Exposure of the macroalga to metals resulted in a disruption of intracellular monovalent cation composition. Intracellular potassium was irreversibly lost and sodium was accumulated by cadmium- or copper-treated U. lactuca, which was assumed to indicate irreversible disruption of the plasmalemma. Exposure to zinc caused an increase in sodium concentrations, whereas potassium concentrations were not significantly different from the controls, suggesting that the integrity of the plasmalemma had been maintained at the zinc concentration used. Intracellular magnesium was also lost from copper-treated algae, which again indicated a loss of integrity of the cell membrane.     

A novel three-stage seaweed (Ulva lactuca) biofilter design for integrated mariculture

Seaweed biofilters have proven their usefulness in the treatment of fishpond effluents. However, their performance poses a dilemma: TAN (Total Ammonia N) uptake rate – and with it seaweed yield and protein content – is inversely proportional to TAN uptake efficiency. The ideal for a seaweed biofilter performance would be a high uptake rate together with high uptake efficiency. The novel three-stage seaweed biofilter design described here has solved this dilemma. The design used the finding that the performance of seaweed ponds depended on the flux of TAN through them, and that therefore effluents with reduced TAN concentration could provide the seaweed with a high TAN flux if the water flow increased proportionally. Effluents from a seabream fishpond were passed through a series of three successively smaller (25, 12.5 and 6.25 m², respectively) air-agitated Ulva lactuca ponds. The diminished inflow TAN concentrations to the second and third ponds of the biofilter system were compensated for by the increased water exchange rates, inversely proportional to their sizes. The biofilter performance was evaluated under several TAN loads. TAN was efficiently removed (85–90%), at a high areal rate (up to 2.9 g N m^-2 d^-1) while producing high protein U. lactuca (up to 44% dw) in all three stages, although with mediocre yields (up to 189 g fresh m^-2 d^-1). Performance of each seaweed biofilter pond correlated not with TAN concentration, but with areal TAN loads. The novel three-stage design provides significant functional and economic improvements in seaweed biofiltration of intensive fishpond water.     

Cell wall structure of the agarophytes Gracilaria tikvahiae and G. cornea (Rhodophyta) and penetration by the epiphyte Ulva lactuca (Chlorophyta)

Use of light, transmission, and scanning electronmicroscopes revealed that the epidermal cell wall ofthe red algal agarophytes Gracilaria tikvahiaeMcLachlan and G. cornea J. Agardh consists of adecklamelle and outer and inner wall layers. The twospecies differed, with G. cornea having asignificantly thicker outer wall and a more diffusedecklamelle. After induction, the zooids of Ulvalactuca would attach to glass slides and the twospecies of Gracilaria via an adhesion pad. Within a few days, 3–5 celled germlings penetrated thedecklamelle and outer wall layer of both basiphytes. By the time the epiphyte germlings reached the 15celled stage, they had penetrated the inner walllayer. The differences in epidermal cell wallconstruction between the two basiphytes may play arole in the ability of zooids of U. lactuca toattach in nature where epiphytization of G.cornea is infrequent.     

Copper-induced intracellular calcium release requires extracellular calcium entry and activation of L-type voltage-dependent calcium channels in Ulva compressa

The marine alga Ulva compressa exposed to 10 µM copper showed a triphasic increase of intracellular calcium with maximal levels at 2, 3 and 12 h involving the activation of ryanodine-, Ins(1,4,5)P₃- and NAADP-sensitive calcium channels. In order to analyze the requirement of extracellular calcium entry for intracellular calcium release as well as the activation of voltage-dependent calcium channels (VDCC) and phospholipase C, U. compressa was treated with EGTA, a non-permeable calcium chelating agent, with verapamil, nipfedipine and diltiazem, inhibitors of L-type VDCC, and with neomycin and U731222, inhibitors of phospholipase C. The release of intracellular calcium was partially inhibited with EGTA at 2 and 3 h and completely inhibited at 12 h of copper exposure and decreased with inhibitors of L-type VDCC and phospholipase C. Thus, copper-induced intracellular calcium release depends on calcium entry and activation of L-type VDCC and phospholipase C. An integrative model of copper-induced cellular responses in U. compressa is presented.     

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.     

Preparation of protoplasts from the green alga Enteromorpha intestinalis (L.) Link

Protoplasts have been obtained from vegetative thallus of the green seaweed Enteromorpha following enzymic digestion with driselase and pectinase. The viability of purified protoplast fractions was assessed by staining and measurements of O₂ uptake and evolution.Abbreviations MES 2-(N-morpholino) ethanesulphonic acid - TES N-tris(hydoxymethyl) methyl-2 aminoethanesulphonic acid     

The effect of water flow on photosynthetic processes of the alga Ulva lactuca L.

Photosynthetic rates of aquatic macrophytes are affected by the diffusion of carbon through the boundary layer which decreases with increasing flow velocities. This is shown by a significant increase in photosynthetic rates of Ulva lactuca fronds exposed to friction velocities (u _*) between 0.0 and 0.3 cm s^–1. No further increase in photosynthetic rates is observed at u _* values between 0.3 and 1.0 cm s^–1. Stagnating flow conditions at saturating light levels caused a 29% reduction in photosynthetic rates. Experiments in a closed system indicate that Ulva may utilize internal carbon sources during periods of stagnation. On a subtidal rock directly exposed to wave action, Ulva lactuca is not exposed to such conditions while in a wave flushed intertidal pool stagnation occurs during very short time intervals based on in situ measurements of u _* levels on fronds of this species using a microprocessor-controlled, autonomous recorder and hot-film sensors.     

Phylogeographical structure, distribution and genetic variation of the green algae Ulva intestinalis and U. compressa (Chlorophyta) in the Baltic Sea area

The marine algae Ulva intestinalis and U. compressa are morphologically plastic with many overlapping characters and are therefore difficult to distinguish from each other. The present distribution of U. intestinalis and U. compressa is investigated along the salinity gradient in the Baltic Sea area through analyses of internal transcribed spacer (ITS) sequence data. Also, the amount and distribution of intraspecific genetic polymorphism in the ITS region is studied allowing inferences on the phylogeographical pattern and postglacial recolonization of the Baltic Sea area. The data show that of the two species only U. intestinalis occurs in the Baltic Sea. The distribution of U. compressa is more restricted than previously reported, and it was not found in salinities lower than 15 ppt. All of Scandinavia and the Baltic Sea were covered with ice during the last ice age and the organisms in the Baltic Sea must have colonized the area after the ice had started to melt. The genetic diversity of U. intestinalis and U. compressa in the Baltic Sea and the neighbouring area was found to be reduced compared to that in the British Isles. This reduction may be the result of either a historical reduction of diversity or an adaptation of specific clones to the northern environmental conditions.     

Gracilaria conferta and its epiphytes: (2) Interrelationship between the red seaweed and Ulva cf. lactuca

Ulva cf. lactuca has been a disturbing competitor of experimental Gracilaria conferta outdoor cultures in Israel. The effect of environmental conditions on the competitive ability of Ulva versus Gracilaria, and the main limiting factors for which these seaweeds compete, were studied here. Single and biculture experiments of both seaweeds showed that Ulva outgrew and damaged Gracilaria under all irradiance and temperature combinations. The higher competitive ability of Ulva cf. lactuca in bicultures was not a result of responses to shading or nitrogen shortage, but rather to a shortage of available inorganic carbon, an increase in pH and apparent excretions of Ulva which inhibited the growth of Gracilaria.     

Ulva and Enteromorpha as indicators of heavy metal pollution

We studied the use of two genera of green macroalgae, Ulva and Enteromorpha, as indicators of heavy metal contamination on the northwest coast of Spain. We evaluated the extent of external contamination by fine particles adhering to the algal thallus and found that although not a problem in Ulva, it may be in Enteromorpha samples. The mean levels of metals in both seaweeds were in accordance with previously reported levels in different species of the genera in clean areas. A large number of significant correlations between levels of metals in macroalgae and in sediment (for both the total and labile fractions) were found. However, almost all of the coefficients of correlation decreased after geochemical normalization of sediment metal concentrations.     

Development of a transplantation technique of Cystoseira amentacea var. stricta and Cystoseira compressa

On the North-Western Mediterranean coastline, the genus Cystoseira (Fucales) has a key role on upper infralittoral community structure. Cystoseira amentacea var. stricta and C. compressa are the most common superficial Cystoseira species. In this area, the coastline is subjected to a strong anthropogenic pressure which has caused severe Cystoseira population decreases. The present study consists in the development of a cheap and easy to use transplantation technique of C. amentacea var. stricta and C. compressa in order to either restore former natural populations or help their settlement on artificial substrates. The thalli were fixed with epoxy glue in holes made by a drill. The success of the technique was assessed by counting the number of remaining transplanted thalli after 3 weeks, 3 months and 6 months. Results were found encouraging with 75% of survival for both species after 6 months. Moreover fertile fronds were observed on the transplanted thalli showing the harmlessness of the technique.     

Copper accumulation, synthesis of ascorbate and activation of ascorbate peroxidase in Enteromorpha compressa (L.) Grev. (Chlorophyta) from heavy metal-enriched environments in northern Chile

Enteromorpha compressa is the dominant species in coastal areas of northern Chile receiving copper mine wastes. Copper remains as the main heavy metal in these coastal waters and it is accumulated in E. compressa growing at the impacted sites. Algae from these sites showed higher levels of lipoperoxides than from non‐impacted sites, which suggests the occurrence of cellular damage resulting from oxidative stress. The strong activation of ascorbate peroxidase detected in this study probably occurs in order to buffer this oxidative stress. Unexpectedly, the activity of glutathione reductase, normally coupled to ascorbate peroxidase activity, was not affected by the chronic exposure to the mine wastes. Moreover, catalase, dehydroascorbate reductase and glutathione peroxidase, commonly reported to buffer oxidative stress in plants and algae, were not detected in E. compressa from any of the studied sites. Levels of total glutathione and phenolic compounds decreased in algae from mine‐impacted sites. In contrast, high levels of dehydroascorbate were found in algae from impacted sites, whereas ascorbate remained unchanged. Therefore, it is suggested that E. compressa tolerates a copper‐enriched environment, and the accompanying oxidative stress, through the accumulation of copper, activation of ascorbate peroxidase, synthesis of ascorbate (accumulated as dehydroascorbate) and consumption of glutathione and water‐soluble phenolic compounds.     

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.     

Synchronization of protoplasts from Glycine max (L.) Merr. and Brassica napus (L.)

Cells of Glycine max originating in a suspension culture and cells of Brassica napus prepared from hypocotyls were synchronized. Synchronization was achieved by preparing protoplasts in the usual way and subsequently letting the protoplasts regenerate into cells by removing the cell-wall-digesting enzymes. More than 70% of the cells had divided synchronously at the end of the first cycle as determined by the mitotic index. The high frequency of mitosis critically depended on the osmolality of the medium. The duration of the S-phase was estimated by measuring the activity of thymidylate kinase as well as incorporation of [³H]deoxythymidine into acid-insoluble material. The data indicate that synchronization is induced by resetting the cell cycle.Abbreviations dTMP deoxythymidine 5-monophosphate - TCA trichloroacetic acid     

Plant regeneration from protoplasts of Enteromorpha intestinalis (Chlorophyta, Ulvophyceae) as seedstock for macroagal culture

A continuous micropropagation was established from protoplasts of thegreen alga Enteromorpha intestinalis. The effects of two differentcrude enzymes and the osmolarity at different concentrations of the enzymesolution on algal protoplast yields were tested. The optimal enzymecomposition for cell wall digestion and protoplast viability was 2%cellulase R 10 Onozuka and 2% Aplysie with 0.5 m mannitol. Largenumbers of Enteromorpha protoplasts were released (10.0 × 10⁶protoplasts from 1 g fresh thalli) and settled on a rangeof substrata. Regeneration of the protoplasts followed the normal patternfor this species. Conditions for pure cultures and efficient systems offloating supports with nets were determined to optimise the product qualityof plantlets of Enteromorpha. A promising storage process has beendeveloped which involves including protoplasts in beads of alginic acid gel.Plants regenerated from protoplasts may also be used as seedstock tofacilitate propagation for macroalgal culture.     

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.