cDNA cloning of a lectin-like gene preferentially expressed in freshwater from the macroalga Ulva limnetica (Ulvales, Chlorophyta)

The macroalgal species Ulva limnetica Ichihara et Shimada was investigated to understand the molecular mechanism of its tolerance or adaptation to freshwater. We detected a 19 kDa protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which accumulated in greater amounts in freshwater conditions compared with marine conditions. The band was excised and the partial amino acid sequence was determined by Edoman degradation. Based on the sequences, we isolated the corresponding cDNA by the rapid amplification of cDNA ends (RACE) technique. The constructed, full-length cDNA was 1272 bp in length, consisting of 198 bp 5'-non-coding region, an open reading frame of 840 bp (279 amino acids), 233 bp 3'-non-coding region and poly (A) tail. The protein encoded by the cDNA showed 30% identity and 45% similarity to lectin isolated from Ulva pertusa Kjellman, and we named this gene ULL (encoding Ulva limnetica lectin-like protein). Northern blot analysis demonstrated that the expression level of the ULL in the freshwater-cultured sample was higher than in the seawater-cultured sample.     

Freshwater population of Ulva flexuosa (Ulvaceae,Chlorophyta) as a food source for great pond snail: Lymnaea stagnalis (Mollusca,Lymnaeidae)

Cosmopolitan species of the genus Ulva (Ulvaceae; Chlorophyta) that populate the littoral zone of marine habitats constitute a staple diet for a variety of organisms, particularly snails, shellfish, polychaetes, and birds. Occurrence of Ulva species (e.g., U. flexuosa and U . prolifera) has also been observed in freshwater inland ecosystems that have no contact with saline water. However, the influence of the development of macroalgal mats of Ulva on indigenous organisms in limnic ecosystems has not been established. This study investigates the trophic relationships between Ulva flexuosa and one species of snail from freshwater habitats in central Europe. During the summer, the great pond snail (Lymnaea stagnalis) consumed Ulva as a source of nutrition even when other algae and plants were available. Lymnaea stagnalis consumed an average of 100 mg of Ulva thalli per day. This level of biomass exceeded the consumption of an alternative food source, the shoots of Elodea canadensis. Ulva thalli are more actively consumed by great pond snails than Elodea shoots, and this is expressed in terms of the differences of biomass consumption. It was also observed that the interior of the monostromatic tubular thalli of Ulva flexuosa serves as a protective shelter for juvenile great pond snails.     

A new green-tide-forming alga, Ulva ohnoi Hiraoka et Shimada sp. nov. (Ulvales, Ulvophyceae) from Japan

Ulva ohnoi Hiraoka et Shimada sp. nov. (Ulvales, Ulvophyceae) is described from southern and western Japan and is characterized by the following combination of features: (i) the large, fragile, easily torn thalli, which are 30–55 μm thick in the upper and middle regions and often have microscopic marginal teeth; (ii) the production of zoids in the upper marginal region; (iii) a regular alternation of dioecious gametophytes and a sporophyte; (iv) the production of free‐floating thalli from torn‐off attached thalli, which reproduce vegetatively by fragmentation and form green tides in summer to autumn; (v) disorderly arranged cells that are polygonal or quadrangular in the upper and middle regions; and (vi) the chloroplast covering the outer face of cell, with 1–3 pyrenoids. Ulva ohnoi differs from U. armohcana Dion et al., U. fasciata Delile, U. reticulata Forsskal, U. scandinavica Eliding and U. spiulosaOkamura et Segawa, which all possess microscopic marginal serrations, in thallus shape, cell shape or life history pattern. It is also distinguished from morphologically similar species by sequences of the nuclear encoded internal transcribed spacers and the 5.8S ribosomal RNA gene and the plastid encoded large subunit of ribulose‐l,5‐bisphosphate carboxylase/oxgenase gene. Furthermore, crossing tests demonstrate that there is a reproductive boundary between U. ohnoi and the most closely related species, U. fasciata and U. reticulata.     

Two modes of bicarbonate utilization in the marine green macroalga Ulva lactuca

The green marine macroalga Ulva lactuca L. was found to be able to utilize HCO₃^? from sea water in two ways. When grown in flowing natural sea water at 16°C under constant dim irradiance, photosynthesis at pH8.4 was suppressed by acetazolamide but unaffected by 4,4′-diisothiocyanostilbene-2,2′-disulphonate. These responses indicate that photosynthetic HCO₃^? utilization was via extracellular carbonic anhydrase (CA) -mediated dehydration followed by CO₂ uptake. The algae were therefore described as being in a ‘CA state’. If treated for more than 10 h in a sea water flow-through system at pH9.8, these thalli became insensitive to acetazolamide but sensitive to 4,4′-diisothiocyanostilbene-2,2′-disulphonate. This suggests the involvement of an anion exchanger (AE) in the direct uptake of HCO₃^?, and these plants were accordingly described as being in an ‘AE state’. Such thalli showed an approximately 10-fold higher apparent affinity for HCO₃^? (at pH9.4) than those in the ‘CA state’, while thalli of both states showed a very high apparent affinity for CO₂. These results suggest that the two modes of HCO₃^? utilization constitute two ways in which inorganic carbon may enter the Ulva lactuca cells, with the direct entry of HCO₃^?, characterizing the ‘AE state’, being inducible and possibly functioning as a complementary uptake system at high external pH values (e.g. under conditions conducive to high photosynthetic rates). Both mechanisms of entry appear to be connected to concentrating CO₂ inside the cell, probably via a separate mechanism operating intracellularly.     

Ulva flexuosa (Ulvaceae,Chlorophyta) inhabiting inland aquatic ecosystems: molecular,morphological and ecological discrimination of subspecies

Species of Ulva (Ulvaceae, Chlorophyta) are relatively common macroalgae in most coastal areas. In this paper, we examine Ulva in inland aquatic ecosystems to determine relationships between the chemical parameters of the water and the Ulva taxa present. Two groups of samples of freshwater Ulva were selected from: (i) habitats with high salinity levels, and (ii) habitats with fresh or slightly saline waters. We showed that these two groups of Ulva, which occupy distinct habitats, represent two separate taxa. The first group represents Ulva flexuosa subsp. pilifera (Kützing) M.J. Wynne, which is very common and regularly encountered in fresh waters, but is equally well represented in slightly saline waters. The second is U. flexuosa subsp. paradoxa (C. Agardh) M.J. Wynne, which is less common, preferring mainly salt-wedge saline waters. We did not find both Ulva flexuosa subspecies together at any of the research sites.

There were significant morphological differences between the Ulva flexuosa subspecies. Ulva flexuosa subsp. pilifera had longer and wider thalli and wider cells than those of subsp. paradoxa. Ulva flexuosa subsp. paradoxa cells were longer and of greater circumference than those of subsp. pilifera. Analyses of the rRNA ITS region and partial rbcL gene sequences demonstrated minor sequence variation within the two subspecies. The results of this study provide detailed information on the characteristics of limnic Ulva habitats and indicate the niches where such populations are likely to occur.     

The induction of reproductive cell formation of Ulva pertusa Kjellman (Ulvales, Ulvophyceae)

Synchronous zooid formation in Ulva pertusa Kjellman was induced in excised disks maintained in sterilized seawater at 20°C, 12:12 h L:D cycle and fluorescent light at 100 μmol photons m ²s ¹. Zooids were reieased from mature disk tissue on the morning of the second or the third day after excision. The degree of zooid formation was found to be dependent on disk size and the region of the mother thalius from which the disk tissue was excised. Zooid formation was induced in more than 90% of small disks (0.9 mm in diameter) which were taken from the margins of the Ulva thalli. When disks were incubated together with a perforated mother thalius, the disks remained sterile. The presence of maturation inhibitors in vegetative thalli is suggested.     

Asexual life history by quadriflagellate swarmers of Ulva spinulosa (Ulvales, Ulvophyceae)

This is the first report of a Ulva species reproducing asexually solely by quadriflagellate swarmers. Ulva spinulosa Okamura et Segawa specimens were collected from Ukibuchi on the Pacific coast of Kochi Prefecture, southern Japan. Quadriflagellate swarmers were released from these specimens. The swarmers showed negative phototaxis before settlement. Thalli cultured from these swarmers also released quadriflagellate swarmers in culture. Microspectrophotometric studies demonstrated equivalent DNA in nuclei of vegetative cells in thalli of U. spinulosa and in sporo‐phytes of the other Ulva species with sexual life history (U. fasciata Delile). Furthermore, the quadriflagellate swarmers of U. spinulosa had the same DNA value, demonstrating that the quadriflagellate swarmers are produced without meiosis.     

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.     

Growth and Thallus Morphogenesis of Ulva mutabilis (Chlorophyta) Depends on A Combination of Two Bacterial Species Excreting Regulatory Factors

Axenic Ulva mutabilis gametes develop parthenogenetically into callus‐like colonies consisting of undifferentiated cells without normal cell walls. From the accompanying microbial flora of established laboratory strains of U. mutabilis with normal morphology, a Roseobacter, a Sulfitobacter, and a Halomonas species were isolated. Each of these microbe species alone induced the development of the Ulva gametes into thalli composed of differentiated cells with characteristic deficiencies. Typical traits of these thalli were: an enhanced rate of cell division not followed by cell expansion, the presence of unusual cell wall protrusions, and the absence of differentiated rhizoid cells. The addition of a Cytophaga species, also derived from the same microbial flora, to either one of the three other strains resulted in the development of normal fast growing thalli with the typical morphology of the algal strain used. These effects are mediated by specific regulatory factors that are excreted into the environment by the bacteria and could be also isolated from the bacterial cell extracts. In contrast with the Cytophaga‐factor, the regulatory factor of the three other bacterial species was also found intracellularly in other bacterial strains not associated with Ulva, but in this case it was not excreted. Functionally, the Roseobacter‐, Sulfitobacter‐, and Halomonas‐factors resemble a cytokinin, while the Cytophaga‐factor acts similar to auxin. Neither factor could be replaced by known phytohormones. The Roseobacter species exhibits a specific chemotactic affinity to the rhizoid cells of U. mutabilis and seems to cooperate with the Cytophaga strain and the alga by chemical communication forming a symbiotic tripartite community.     

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.     

Three species of Ulva (Ulvophyceae) from the Persian Gulf as potential sources of protein,essential amino acids and fatty acids

Three species of the cosmopolitan genus Ulva (U. paschima , U. chaugulii and U. ohnoi ) from the Persian Gulf were identified using morphological and molecular markers, and were analyzed for total lipids, fatty acids, proteins and amino acid profiles. Our results show that the studied Ulva species have high protein content (9?25% dry weight), contain essential fatty acids and nearly all essential amino acids. This suggests Ulva could have potential as a form of human nutrition and animal feed with a low carbon footprint.     

Effect of Temperature and Illumination on Growth and Reproduction of the Green Alga Ulva fenestrata

The combined effect of temperature (5, 10, 15, and 20°C) and illumination (40 and 60 mE/(m² s)) on growth and reproduction of the green marine alga Ulva fenestrata P. et R. from the sublittoral zone of Amursky Bay, Sea of Japan, was studied in the laboratory environment in the months April–July, 2000. It was demonstrated that the temperature of 5°C and illumination of 40 mE/(m² s) are the most favorable for maintaining the vegetative mass of the algae. A water temperature of 10°C and illumination of 40 mE/(m² s) are the optimum conditions for vegetative growth of U. fenestrata thalli. A temperature decrease and increase by 5°C reduces the growth rate on average by 30%. Sporo- and gametogenesis in U. fenestrata are the most regular (every 10 days) and occupy the greatest disk area at a water temperature of 15°C and illumination of 40 mE/(m² s). Vegetative growth of thalli is sharply inhibited at the stage of cell preparation to gametogenesis a day before the beginning of gamete formation.     

Growth of the seaweed Ulva rigida C. Agardh in relation to biomass densities, internal nutrient pools and external nutrient supply in the Sacca di Goro lagoon (Northern Italy)

Growth of the seaweed Ulva rigida C. Agardh was investigated in relation to biomass densities, internal nutrient pools and external nutrient supply. Research was carried out from 23 March to 5 July 1994 in the Sacca di Goro (Po Delta, Northern Italy), whose south-eastern part was covered by extensive mats of Ulva rigida. Two types of field experiments were conducted by incubating Ulva thalli inside large cages. In the first experiment, beginning on 23 March, 100 g of wet thalli were placed into the cages, allowed to grow for two weeks, then collected and replaced. This procedure was repeated 8 times over the study period. In the second experiment, Ulva thalli were left inside the cages and collected at selected time intervals (14, 27, 41, 64 and 76 days) in order to simulate the effects of increased density on growth and nutrient storage.We recorded specific growth rates (NGR) ranging from 0.025 to 0.081 d^–1 for a period up to two months in the repeated short-term experiments performed at relatively low initial algal densities (300–500 g AFDW m^–3). These NGR resulted significantly related to dissolved inorganic nitrogen (DIN) in the water column. Tissue concentrations of total Kjeldahl nitrogen (TN) were almost constant, while extractable nitrate decreased in a similar manner to DIN in the water column. Total phosphorus showed considerable variation, probably linked to pulsed freshwater inflow.In the long-term incubation experiment, NGR of Ulva was inversely related to density. Internal concentrations of both total P and TN reached maximum values after one month; thereafter P concentration remained almost constant, while TN decreased below 2% w/w (by dry weight). The TN decrease was also accompanied by an abrupt decrease in nitrate tissue concentration. The biomass incubated over the two month period suffered a progressive N limitation as shown by a decreasing NY ratio (49.4 to 14.6). The reciprocal control of Ulva against biogeochemical environment and viceversa is a key factor in explaining both resource competition and successional stages in primary producer communities dominated by Ulva. However, when the biomass exceeds a critical threshold level, approximately 1 kg AFDW m^–3, the macroalgal community switches from active production to rapid decomposition, probably as a result of selfshading, biomass density and development of anaerobic conditions within the macroalgal beds.     

Response of seedling communities to mammalian pest eradication on Ulva Island, Rakiura National Park, New Zealand

Norway rats (Rattus norvegicus) were eradicated from Ulva Island, Rakiura National Park, in 1996. The aim of our work was to determine if seedlings and saplings increased in density and/or species richness following this eradication. In 2003, we took advantage of eight permanent plots (5 × 5 m) that had been established on Ulva Island in 1991, by counting seedlings and saplings of woody species, including tree ferns. Over this period, total numbers of woody seedlings (< 30 cm tall), and saplings (30 cm – 2 m tall) did not increase significantly (P > 0.05). However, seedlings of two species, Dicksonia squarrosa and Pittosporum colensoi, and saplings of one species, Coprosma colensoi, did increase in numbers significantly. Little change in the species richness of seedlings or saplings was recorded. The removal of Norway rats may have been partly responsible for the recorded increases in density, though the eradication of white-tailed deer (Odocoileus virginianus borealis) from Ulva Island approximately two decades prior to this work is an important confounding factor.     

孔石莼(Ulva pertusa)凝集素的分离纯化及性质的研究

为抑制肿瘤细胞增殖和防治有关病害提供基础理论依据 ,将孔石莼 (Ulva pertusa)经磷酸盐缓冲液抽提 ,2 0 %～ 75%硫酸铵分级沉淀 ,牛甲状腺球蛋白 - Sepharose4B亲和层析 ,可以从绿藻孔石莼中纯化出孔石莼凝集素 (UPL) ,在 PAGE上显示单一蛋白染色带 ,在等电聚焦电泳上显示单一蛋白染色带 ,其 p I为 8.40 .纯化后的 UPL的最大紫外吸收峰在 2 85nm,用 Sephadex G- 2 0 0分子筛层析测得其分子量为 1 1 0 4 7.该凝集素可以凝集人的 A、B、AB、O型红细胞 ,且凝集活性相同 ,在对人 (A、B、AB、O)兔、鲤、鲫的红细胞的凝集作用中 ,兔的凝集作用最强 .该凝集素凝集兔红细胞的作用不被 D-半乳糖、D-果糖、葡萄糖、蔗糖、甘露聚糖、γ球蛋白、卵清蛋白所抑制 ,仅被牛甲状腺球蛋白抑制 ,最小抑制浓度为 6.2 0 g/L.该凝集素在 p H4.0～ 1 0 .1 4范围内均有活性 ,但在p H6.50～ 9.51范围内活性较高 ,该凝集活性在 85℃加热 1 h,活力仍未改变 ,说明具有很强的耐热性 .     

Crossing test among floating Ulva thalli forming `green tide' in Japan

Crossing tests were made to determine the relationship between the identified Ulva pertusa, which commonly grows in Japan as an attached form on exposed rocks, and the floating Ulva forming "green tide" inside calm bays. The floating Ulva thalli were collected from five major green tide sites in Japan (Yokohama, Mikawa, Miyajima, Kochi and Hakata). Reproductive maturation was induced in U. pertusa and the floating thalli from each site. Mating between induced gametes was observed. It is therefore believed that the floating thalli from Yokohama, Mikawa and Miyajima were mainly U. pertusa, while those from Kochi and Hakata were of a different species (Ulva sp.1). Furthermore, the Ulva species found in Mikawa is also a species (Ulva sp.2) different from both U. pertusa and Ulva sp.1.     

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.     

Effects of ocean acidification on growth and physiology of Ulva lactuca (Chlorophyta) in a rockpool‐scenario

Rising atmospheric CO₂‐concentrations will have severe consequences for a variety of biological processes. We investigated the responses of the green alga Ulva lactuca (Linnaeus) to rising CO₂‐concentrations in a rockpool scenario. U. lactuca was cultured under aeration with air containing either preindustrial pCO₂ (280 μatm) or the pCO₂ predicted by the end of the 21st century (700 μatm) for 31 days. We addressed the following question: Will elevated CO₂‐concentrations affect photosynthesis (net photosynthesis, maximum relative electron transport rate (rETR(max)), maximum quantum yield (Fv/Fm), pigment composition) and growth of U. lactuca in rockpools with limited water exchange? Two phases of the experiment were distinguished: In the initial phase (day 1–4) the Seawater Carbonate System (SWCS) of the culture medium could be adjusted to the selected atmospheric pCO₂ condition by continuous aeration with target pCO₂ values. In the second phase (day 4–31) the SWCS was largely determined by the metabolism of the growing U. lactuca biomass. In the initial phase, Fv/Fm and rETR(max) were only slightly elevated at high CO₂‐concentrations, whereas growth was significantly enhanced. After 31 days the Chl a content of the thalli was significantly lower under future conditions and the photosynthesis of thalli grown under preindustrial conditions was not dependent on external carbonic anhydrase. Biomass increased significantly at high CO₂‐concentrations. At low CO₂‐concentrations most adult thalli disintegrated between day 14 and 21, whereas at high CO₂‐concentrations most thalli remained integer until day 31. Thallus disintegration at low CO₂‐concentrations was mirrored by a drastic decline in seawater dissolved inorganic carbon and HCO₃^?. Accordingly, the SWCS differed significantly between the treatments. Our results indicated a slight enhancement of photosynthetic performance and significantly elevated growth of U. lactuca at future CO₂‐concentrations. The accelerated thallus disintegration at high CO₂‐concentrations under conditions of limited water exchange indicates additional CO₂ effects on the life cycle of U. lactuca when living in rockpools.