The effect of localised eutrophication on competition between Ulva lactuca (Ulvaceae,Chlorophyta) and a commercial resource of Gracilaria verrucosa (Gracilariaceae,Rhodophyta)

In spring (August) 1993 a bloom of Ulva lactuca appeared for the first time in Saldanha Bay, South Africa, and persisted through summer. Ulva wash-ups contaminated the beach and part of the commercial Gracilaria beach-cast had to be discarded. The biomass and distribution of Gracilaria and Ulva are described in relation to the seasonal water chemistry of the bay. Gracilaria survives in deeper water in summer by the pulsing of nutrients on an approximately 6-day cycle of movement of the thermocline that separates nutrient-rich bottom water from warm oligotrophic surface water. Ammonium-rich fish-factory discharge into this surface layer in a sector of the bay provided localised conditions for Ulva to out-compete Gracilaria at depths of 2–5m, demonstrating the powerful disruptive effect of eutrophication in this strongly stratified system.     

Determination of the composition of the fibrillar part of Gracilaria verrucosa (Gracilariales,Rhodophyta) cell wall in order to prepare protoplasts

The cell wall of Gracilaria verrucosa is composed of two fractions: a matrix made of agar and a skeleton whose composition is unknown. This fibrillar part was isolated using both physical and chemical techniques. Total hydrolysis followed by gas-liquid chromatography allowed us to establish the sugar composition. Enzymatic degradations were carried out with cellulases, xylanases, agarases and pectinases. Efficiencies of the enzymatic digestions were monitored by both chemical analysis and electron microscopy. Pectinases had no effect. The fibrillar part was composed mainly of a cellulosic network that was unmasked by the xylanase action and degraded after cellulase digestion. The results suggest that a cocktail composed of agarases and cellulases can be used successfully to prepare protoplasts from Gracilaria verrucosa.     

Chemical and physical-chemical characteristics of dietary fibres from Ulva lactuca (L.) Thuret and Enteromorpha compressa (L.) Grev.

Dietary fibres from Ulva lactuca (L.) Thuret (sea lettuce) and Enteromorpha compressa (L.) Grev. (A.O. nori) were measured according to a ‘standard’ method and a ‘physiological’ protocol simulating the gastric and intestinal environments. U. lactuca contained 15.8–8.0% soluble and 24.2–32.6% insoluble fibres according to the ‘standard’ and ‘physiological’ methods, respectively. For E. compressa, these values were 14.9–15.9 and 21.6–28.7%, respectively. For both algae, the composition suggests that the soluble fibres were xylorhamnoglycuronans sulphates and insoluble fibres were essentially composed of glucans. No marked chemical compositional variation was observed between soluble fractions extracted under the simulated gastric and intestinal conditions. Fibres in both algae are hydrophilic but the water holding capacities were higher after extraction of soluble fibres (5.5–9.5 g g⁻¹ for the dry algae; 14.0–16.0 g g⁻¹ for the standard insoluble fibres). Water soluble fibres demonstrated low intrinsic viscosities at 37 °C in buffers, particularly those from E. compressa (36.0–36.5 ml g⁻¹), and was affected by pH for those of U. lactuca (147.5 ml g⁻¹ at pH 3.0 and 175.0 ml g⁻¹ at pH 7.3).     

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.     

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.     

Ultrastructure of the cell wall of Gracilaria cf. verrucosa (Gracilariales,Rhodophyta): effects of steam explosion

Ultrastructural (SEM, TEM) and cytochemical organization of the skeletal and matrix polysaccharides of fresh and steam exploded Gracilaria verrucosa thalli were compared to chemical analyses of the agar extracted after this treatment. Upon steam explosion, surface microfractures appeared in limited areas of the cortex. SEM preparations indicated that apical and medullary regions were the most affected, resulting in detachment of adjacent cells. In TEM sections, the cuticle of the surface layer exhibited deeply altered zones with a spongy appearance. In the cytoplasm starch grains were disrupted. The skeletal component of the immediate cell wall was maintained with a different organization of the microfibrils and was positive to periodic acid-thiosemicarbazide-silver proteinate. The intercellular matrix contained granules and fibrils and lacked a ruthenium red reaction. These results are consistent with lower sulfate-ester and higher glucose contents detected in agars extracted using steam explosion.     

Responses of the macroalga Gracilaria tenuistipitata var. liui (Rhodophyta) to iron stress

Chlorophyll (Chl), phycoerythrin (PE), total nitrogen (TN% dw) and Fein tissues were measured in Fe-deficient cultures of Gracilariatenuistipitata var. liui over a period of 60 days. ⁵⁵Fe uptakeand photosynthetic carbon fixation (NaH¹⁴CO³) werecompared in Fe-rich and Fe-deficient cultures and analyzed the effects ofFe-deficiency on the ultrastructure. The maximum carbon fixationdecreased significantly (p < 0.01) under Fe-deficiency. Thechlorophyll and phycoerythrin contents also declined with decreasing tissueiron content, falling, respectively, to 7.9 and 33.8% of their originallevel. Photosynthesis in Fe-deficient cells became light-saturated at lowerirradiance than the control. Total N in tissue decreased from 3.65 to2.49%. ⁵⁵Fe uptake rate for cultures grown on NO₃^-was measured following resuspension in either NH₄⁺ orNO₃^- as N source. Enhanced Fe uptake developedunder Fe stress, especially with cells resuspended in NH⁺₄-N medium. The Vmaxfor Fe uptake was higher with NH₄⁺ thanNO₃^- (62.8 versus 12.1 pmol mg dw^-1 h^-1). The requirement for N accelerates further Fe uptake. Ultrastructuralobservations of Fe-deficient cells showed reductions in chloroplast number,degeneration of lamellar organization, decrease in mitochondrial matrixdensity and variation in accumulation body number and morphology. During Fe-deficiency, the growth rate continued to decline and after 40days of iron deficiency, no further growth was detectable, and eventuallyiron deficiency resulted in chlorosis. The results suggest that the lowergrowth rate of Gracilaria tenuistipitata var. liui underFe-deficiency may result from largely from inhibition of photosynthesis andnitrogen utilization.     

Production of bioflavor by regeneration from protoplasts of Ulva pertusa (Ulvales,Chlorophyta)

Protoplasts were isolated from thalli of Ulva pertusa using a mixed enzyme solution of 2.0% Cellulase Onozuka R-10, 2.0% Macerozyme R-10, and 2.0% Driselase. Isolated protoplasts regenerated cell walls, developed into thalli, and propagated in large numbers under aeration in the preparative scale-culture system. Typical bioflavor compounds produced from the regenerated plants, as well as from field-collected plants, were found to be long chain aldehydes, which gave a typical seaweed odor. The long chain aldehydes were formed enzymatically from unsaturated fatty acids and released into the culture fluid. A Percoll/mannitol discontinuous density gradient separation of the heterogeneous protoplasts led to a selection of cell lines with high production of bioflavor. The cells that regenerated from protoplasts were immobilized by polymer matrices such as alginate, -carrageenan, agarose, and agar. Living cells entrapped in alginate beads in aerated cultures survived best. However, the beads started to breakdown after two months. The immobilized cells demonstrated a higher bioflavor production than did the cultured cells.     

Optimisation of RNA extraction from Gracilaria changii (Gracilariales, Rhodophyta)

RNA extraction from seaweed tissues is problematic due to the presence of polysaccharides and polyphenolic compounds upon cell disruption. Besides, a successful RNA isolation from seaweed tissues can sometimes be strain- and species-specific. Four different methods were used to extract RNA from Gracilaria changii (Gracilariales, Rhodophyta), collected from the mangrove area at Morib, Selangor, Malaysia. An optimised and modified total RNA extraction method was developed for this recalcitrant species. The use of sand in tissue grinding, and the incorporation of phenol extraction at the initial stage resulted in the highest RNA yield (0.65–1.14 g g^–1 fresh weight) with high quality (A_260:280 ratio 1.80–2.05). The RNA obtained is suitable for cDNA synthesis and future functional genomic studies.     

Viability and dissemination of spermatia of Gracilaria verrucosa (Gracilariales,Rhodophyta)

The dissemination and viability of Gracilaria verrucosa spermatia were tested. Crosses were performed among three males and three females from Cape Gris Nez, northern France. Laboratory experiments show that spermatia have a mean fertile life of about five hours. Field studies show that spermatia are dispersed by stream and tidal currents and that fertilization can occur at least 80 m from a population.     

Chemical characterisation and gelling properties of cell wall polysaccharides from species of Ulva (Ulvales,Chlorophyta)

Two alkali-soluble polysaccharide fractions from the cell wall of Ulva rigida were determined to be ,-1,4-linked glucoxylans and a -1,4-linked glucuronan by chemical and NMR spectroscopic analysis. The ¹³C NMR spectrum of water-soluble xyloglucuronorhamnan sulfate from Ulva rigida referred to as ulvan is reported and the ¹³C and ¹H NMR chemical shifts of its major repeating unit, the aldobiuronic acid -D-GlcA-(1,4)-L-Rha, are given. The composition and gelling properties of ulvan from Ulva species from green tides are also reported. The thermoreversible gel required both calcium and borate ions and the shear storage modulus G was ion concentration dependent. The mechanism of gelation and the associations of the different Ulva cell wall soluble polysaccharides are discussed.     

Polysaccharides from Ulva pertusa (Chlorophyta) and preliminary studies on their antihyperlipidemia activity

Polysaccharides from Ulva pertusa were isolated and prepared by extraction in hot water and precipitation by ethanol. The water-soluble polysaccharides were chemically well defined, containing 47.0% total carbohydrate, 23.2% uronic acids, 17.1% sulfate groups, 1.0% N and 29.9% ash. Gas chromatography analysis demonstrated that the neutral sugars were mainly composed of rhamnose, xylose and glucose and smaller amounts of mannose, galactose and arabinose. The FTIR and ¹³C-NMR spectra indicated that basic repeating units of the polysaccharides were (-D-GlcpA-(1-> 4)--L-Rhap 3S) and (-L-IdopA-(1-> 4)--L-Rhap 3S). Fifty ICR mice were used to study the effect of water-soluble polysaccharides from Ulva pertusa on the level of plasma lipids, with inositol niacinate as positive control. The results indicated that the polysaccharides significantly lowered the contents of plasma total cholesterol, low-density lipoprotein cholesterol, triglyceride and markedly increased the contents of serum high-density lipoprotein cholesterol, compared with the hyperlipidemia control group (p > 0.01). Moreover, administration of polysaccharides significantly decreased the atherogenic index. The present results suggest that the polysaccharides from Ulva pertusa have great potential for preventing ischemic cardiovascular and cerebrovascular diseases.     

Unusual cell structures in tumor-like formations of Gracilaria (Rhodophyta)

This paper deals with electron microscopic observations on cultivated plants of the marine red alga Gracilaria verrucosa which developed simple galls; also sea collected material, without galls, had been studied. The galls showed unusual but characteristic cell structures, caterpillar-like bodies, containing rows of fusiform bodies. These were found mostly in the cytoplasm near the plastids, in one case connected with the endoplasmic reticulum, occasionally even inside the nucleus, and are described here, as far as we know, for the first time. It does not seem probable that the caterpillar-like bodies represent mitochondria or bacteria, but the hypothesis that fusiform bodies are related to virus-like structures is discussed. The normal tissues as well as the gall tissue of the laboratory plants contained, besides plastids typical for the red algae, another type of plastids characterized by tubular thylakoids.This work was supported by grants from Consiglio Nazionale delle Ricerche (Rome) and Ministero dell'Agricoltura e delle Foreste of Italy.     

Differentiation of Male Gametes in Gracilaria verrucosa (Rhodophyta, Gracilariales)

The development of male gametes (spermacia) in the red alga Gracilaria verrucosa has been studied using methods of transmission electron microscopy. Early spermatangia located along the wall of the conceptacle show an elongated shape in the thin sections. In the central part of the electron-dense cytoplasm of these cells there is a nucleus; numerous fibrous vesicles are arranged in the periphery. During the process of differentiation, the spermatangia become more rounded in shape and a large spermatangial vesicle is developed. The subsequent development of spermatium is accompanied by polarization of the spermatangium and the subsequent excretion of the spermatangial vesicle. The spermatia are oval cells containing a nucleus and fibrous vesicles. The process of differentiation of male gametes in G. verrucosa does not differ from that in five species of the genus Gracilaria, where it has already been studied. However, any conclusions about the degree of similarity between the spermatia in all the studied species can be made only after a detailed comparative analysis of the ultrastructural characteristics of these gametes.     

Endogenous and exogenous elicitors of a hypersensitive response in Gracilaria conferta (Rhodophyta)

Certain forms of oligocellulose and certainbacterially excreted peptides were identified asendogenous and exogenous elicitors, respectively, ofa tip bleaching response in Gracilaria conferta(Schousboe ex Montagne) J. et G. Feldmann. Thehalf-maximal tip bleaching response was observed when31.1 M cellobiose or 11.6 M cellotetraosewere present in the growth medium. In contrast, noresponse was detected after exposure to glucose,cellotriose, cellopentaose or maltooligosaccharides.The response was thus strongly dependent on themolecular size of the oligocellulose and onlysaccharides that consisted of an even number ofglucose residues were elicitor-active. Three bacterialspecies that had earlier been identified as potentialinducers of the tip bleaching symptom excretedelicitor-active compounds into the growth medium.These compounds were protease-sensitive and thuspeptides or proteins. The tip bleaching-inducingcompound that was excreted by one Cytophaga-likeorganism was partially purified. It could be extractedfrom culture supernatants with chloroform and itsmolecular size was between 700 and 1500 Da,corresponding with a structure of 4–20 amino acids.Various endogenous and exogenous elicitors are thusrecognized by G. conferta and allow this alga torespond hypersensitively to the maceration of its cellwall skeleton or just to the presence of certainepiphytic organisms.     

Some observations on cell wall structure and taxonomy ofPhymatodocis nordstedtiana (Conjugatophyceae,Chlorophyta)

Filaments ofPhymatodocis nordstedtiana Wolle were isolated from a sample of a Texan lake. Cultures were established and examined by light and scanning electron (SEM) as well as transmission electron microscopy (TEM). It is shown that the pores apparent on light microscopical examination are not of the cosmaroid type as expected. TEM examination disclosed that they are similar to those found in the generaClosterium Ralfs andPenium Bréb. Furthermore, it could be demonstrated by light and SEM microscopy that the primary cell wall is shed during cell division. The remaining secondary cell wall of the mature cell consists of interwoven bands of parallel microfibrils. A conspicuous overlap of the semicell walls clearly denotes the isthmus region. The significance of these deviations unusual for desmids is discussed. Suggestions are made that the taxonomic position ofPh. nordstedtiana should be re-evaluated.     

PCR-identification of Dunaliella salina (Volvocales, Chlorophyta) and its growth characteristics

The saline pond microalga, Dunaliella salina (Dunal) Teod. maintained in De Walne's (basal) medium under laboratory conditions was confirmed by amplifying the chromosomal DNA of the microalga by PCR with specific primers MA1 and MA2. Seaweed extracts obtained from Sargassum wightii and Ulva lactuca were amended separately at 1.0%, 1.5%, 2.0% and 2.5% levels to the basal medium in order to assess their potential on the growth and concentration of pigments, viz. Chl a, Chl b and beta-carotene of the alga. beta-Carotene was isolated and visible absorption spectrum was taken at 443 and 475 nm confirmed the presence of 9-cis-beta-carotene and all-trans-beta-carotene isomers. Maximum yield, highest division rate (mu) and highest pigment concentrations were observed in the cells grown in 1.5% S. wightii and 2.0% U. lactuca amended medium and these cells were subjected to DAPI staining. The results of epifluorescence microscopy and image analysis revealed a significant enhancement of the cell and nuclear area of the microalgae.     

Inorganic nutrition in pond cultivated Gracilaria conferta (Rhodophyta): nitrogen, phosphate and sulfate

The experience gained in the outdoor cultivation system of Gracilariaconferta in Israel suggests three main inorganic variables: inorganic carbon,macronutrients and micronutrients. The effects of the macronutrientsnitrogen, phosphate and sulfate on growth rate, dry weight yield, and agaryield and quality were assessed in this research. The starvation periodbetween ammonium pulse feedings affected the agar yield and its quality asa function of the water temperature. Phosphate was the secondmacronutrient in the usual 1:10 proportion. Dark starvation as well assulfate starvation decreased agar yield. The results of nitrogen or sulfatestarvation suggest a biphasic process of agar biosynthesis. Starting with anoverall growth stage richer with sulfated agarose, and turning into an agaraccumulation stage with more neutral agarose. The elucidation of theintegrated effects of starvation and seasonality on agar production of Gracilaria is the major contribution of this research.