HAPLOID PARTHENOGENETIC SPOROPHYTES OF LAMINARIA JAPONICA (PHAEOPHYCEAE)1

Parthenogenetic sporophytes were obtained from three strains of Laminaria japonica Areschoug. These sporophytes grew to maturity in the sea, producine spores that all grew into female gametophytes. These female gametophytes gave rise to another generation of parthenogenetic sporophytes during the next year, so that by the year 1990 parthenogenetic sporophytes had been cultivated for 12, 9, and 7 generations, respectively, for the three strains. When female gametophytes from parthenogenetic sporophytes were combined with normal male gametophytes, normal sporophytes that reproduced and gave rise to both female and male gametophytes were obtained. The parthenogenetic sporophytes were shorter and narrower than the normal sporophytes of the same strain. Chromosome counts on mature sporophytes showed that normal sporophytes (from fertilized eggs) were diploid (2n = approximately 40) and that the spores they produced were haploid (n = approximately 20), while nuclei from both somatic and sporangial cells in parthenogenetic sporophytes were haploid. All gametophytes were haploid. Young sporophytes derived from cultures with both female and male gametophytes were diploid, while young, sporophytes obtained from female gametophytes from parthenogenetic sporophytes had haploid, diploid, or polyploidy chromosome numbers. Polyploidy was associated with abnormal cell shapes. The presence of haploid parthenogenetic sporophytes should be use in breeding kelp strains with useful characteristics, since the sporophyte phenotype is expressed from a haploid genotype which can be more readily selected.     

EFFECTS OF NITRATE CONCENTRATION ON THE GROWTH AND PHYSIOLOGY OF LAMINARIA SACCHARINA (PHAEOPHYTA) IN CULTURE1,2

Laminaria saccharina Lamour. sporophytes were grown in enriched and synthetic media through a range of nitrate concentrations, There was an approximately linear relationship between growth and nutrient concentration up to 10 μ substrate concentration. The half-saturation constant (K_{2) was ca. 1.4 μ NO3-. The internal levels of NO3- increased at substrate concentrations above 10 μM b>3- and reached levels several thousand times higher than the surrounding medium. Thus there is evidence for luxury consumption of NOsb>3}^-. The chlorophyll content and photosynthetic capacities of plants also increased with increasing external NO₃^- The ecological implications of this work are considered.     

Carbohydrate degradation and methane production during fermentation ofLaminaria saccharina (Laminariales,Phaeophyceae)

Anaerobic digestion of the brown algaLaminaria saccharina (L.) Lamour. harvested in spring and autumn was carried out at controlled laboratory conditions in stirred fermentor systems. Due to the normal seasonal variations, the autumn material had a much higher content of carbohydrates such as mannitol and laminaran. Both batch and semi-continuous feeding conditions were investigated for periods up to 800 h, with inoculum provided from previous kelp fermentations. In batch cultures, the methane yield from the autumn material was doubled compared to that of the spring material. Semi-continuous conditions gave more similar methane yields for both raw materials, 0.22 and 0.27 l CH₄ per g VS for spring and autumn material, respectively. In all experiments, mannitol and laminaran were reduced to less than 5 of the initial values within 24–48 hours after inoculation, whereas 30 of the alginate content was detectable even after 30 days. Viscometry revealed that this material was severely depolymerized, and alginate lyase activity was found to develop rapidly in all cultures. Although mannitol and laminaran were fermented much faster than alginate, the total accumulated methane yields seemed to be determined by the total carbohydrate content of the raw material during extended semi-continuous feeding.(*author for correspondence)     

A simple method for separating cells of Microcystis aeruginosa for counting

Heat was used to break up the mucilage-bound colonies of the blue-green alga, Microcystis aeruginosa Kütz. emend. Elenkin, for cell counting. Samples were heated in a water bath at 80°C for 5–10 min, then agitated in a Vortex mixer for 30–60 s. The treatment separated cells completely and reaggregation of cells did not occur. There was no evidence that heating distintegrated the cells.     

ISOLATION AND CHARACTERIZATION OF SIX cDNAs INVOLVED IN CARBON METABOLISM IN LAMINARIA DIGITATA (PHAEOPHYCEAE)

An expressed sequence tag (EST) approach was used to retrieve cDNA clones involved in carbon metabolism in Laminaria digitata Lamouroux. Six partial open reading frames were identified, respectively encoding an α-type carbonic anhydrase (CA), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), phosphoglycerate kinase (PGK), glycolate oxidase (GLO), and GDP-4-keto-6-d-mannose-3,5-epimerase-4-reductase, also known as fucose synthase (FS). These enzymes were further characterized through Southern blot analyses, amino acid sequence comparisons, and patterns of expression. In contrast to the other genes, which are expressed in both the gametophytic and sporophytic generations of L. digitata, the α-type carbonic anhydrase messenger RNA was shown by RT-PCR and northern blot analyses to be present in the gametophytes only. The evolutionary relationships of these genes and their interest as molecular tools for investigating carbon fluxes in brown algae are discussed.     

NITRATE UPTAKE BY LAMINARIA LONGICRURIS (PHAEOPHYCEAE)1,2

Laminaria longicrucis De la Pylaie took up exogenous nitrate under both summer and winter conditions. During July and August no NO₃^- was detected in the ambient water or in algal tissues although it was present in both in February. Discs (2.3 cm diam.) of thin blade tissue were incubated with NO₃^- at four temperatures, with and without illumination. Similar values Jor NO₃^- uptake were found for both summer and winter collected plants when measured in light at 0 C. An apparent K of 4–6 μM was recorded for both types of plants; the V_max ranged from 7 to 10 μmol h^-1 g^-1 dry wt measured in ca. 1800 μW cm^-2 of cool-white fluorescent light. Uptake rates at 5 C were 66%, and at 0 C 30% of those for controls run at 15 C. The alga scavenged NO₃- from solutions <0.5 μM. Ammonia did not inhibit NO₃- uptake. Antibiotic pretreatment reduced NO₃- uptake by a maximum of 12%. Nitrite uptake was inhibited in proportion to the concentration of NO₃- in the medium.     

Photolithotrophic cultivation of <Emphasis Type="Italic">Laminaria japonica</Emphasis> gametophyte cells in a silicone tubular membrane-aerated photobioreactor

Gametophyte cells of brown algae Laminaria japonica were employed both in a modified silicone tubular membrane-aerated photobioreactor (bubble-less cultivation mode) and a bubble-column photobioreactor (bubbling cultivation mode), to study different gas–liquid mixing modes on cell growth rate and cell physiological status. With an inoculum density of 50 mg DCW l⁻¹, in modified artificial Pacific seawater (APSW) medium at 13°C, light intensity of 60 μE m⁻² s⁻¹, light cycle of 16/8 h L/D, and aeration rate of 60 ml min⁻¹, the specific growth rates were 0.082 d⁻¹ for bubble-less mode and 0.070 d⁻¹ for bubbling mode with biomass, in the form of dry cell density, increasing 10.9 and 6.8 times, respectively, during the 36 days’ photolithotrophic cultivation. The specific oxygen evolution rate under bubble-less mode was 39.6% higher than under bubbling mode on the 18th day. The gametophyte cells grew in cell aggregates with clump sizes, at day 36, of 1.5 mm and 0.5 mm diameter under bubble-less and bubbling mode respectively and cell injury percentages of 5.1% and 21.1%, respectively. The silicone tubular membrane-aerated photobioreactor was better suited for the cultivation of fragile macroalgal gametophyte cells due to the absence of hydrodynamic shear stress caused by fluid turbulence and the presence of a bubble-less gas supply.     

AN ALTERNATE GROWTH PATTERN FOR LAMINARIA LONGICRURIS1,2

A population of Laminaria longicruris de la Pylaie was followed for a year at Bic Island, Quebec, Canada where nutrient levels in the seawater were elevated throughout the year. Tagged kelp were measured each month for growth and analyzed for alginic acid, laminaran, mannitol, carbon, nitrogen, and nitrate. Maximum growth (3.5 cm · d^?1) was observed in June, and minimal growth (0.18 cm · d^?1) from December to February, when ice cover limited light levels. No reserves of carbon or nitrate were formed. Laminaran levels remained below 2.7% dry weight while tissue nitrate did not exceed 0.75 μmol · g^?1 dry weight. Total carbon produced per plant was 40 g C · yr^?1. Nutrient availability enables the kelp to take advantage of summer light and temperature conditions to grow rapidly.     

Differences in resource storage pattern between Laminaria longissima and Laminaria diabolica (Laminariaceae; Phaeophyta) reflecting their morphological characteristics

The kelps Laminaria longissima and L. diabolica, belonging to the groups of L. angustata and L. japonica, respectively, differ greatly in their morphological characteristics although their geographical distributions overlap widely along the eastern coast of Hokkaido. To clarify the interaction between the morphological and physiological characteristics of the two species, and their link with environmental variables, hatchery-raised young sporophytes of L. longissima and L. diabolica collected from Hokkaido were cultivated simultaneously under similar conditions in Matsushima Bay, Miyagi, from January to July 2004. Seasonal morphological characteristics, gross photosynthetic rate, nutrient uptake rates, and resource contents were examined. The blade lengths of L. longissima and L. diabolica reached a maximum of 329.9 cm and 256.7 cm, respectively, in April to May, and decreased to 284.4 cm and 68.6 cm, respectively, in July. The total elongation length of L. longissima (412.5 cm) was similar to that of L. diabolica (373.8 cm). However, the total erosion length of L. longissima (145.9 cm) was approximately half that of L. diabolica (302.9 cm). The gross photosynthetic rate and uptake rates of NH₄-N, NO₃-N, and PO₄-P of the two species were similar. However, the carbon, nitrogen, and phosphorus contents were transferred and stored in the whole blade tissues in the case of L. longissima, but in the meristem of L. diabolica from May to June. These results suggest that morphological differences are a response to different resource storage patterns. The storage patterns of L. longissima and L. diabolica are likely to be genetically fixed characteristics, which have evolved in adaptation to the specific habitat environments of the groups of L. angustata and L. japonica. The low water temperature and rich nutrients provided by the Oyashio Current are conducive to storage of resources in the whole blade tissues and a large surface area retained for photosynthesis and nutrient uptake in the L. angustata group. Conversely, high temperature and poor nutrients, or large fluctuations in these parameters, provided by the Tsushima Warm Current are more conducive to intensive storage of resources in the meristem for maturation and further growth in the L. japonica group. L. diabolica retains the storage pattern of the L. japonica group but grows in regions affected by the Oyashio Current, allowing it to become the widest distributed Laminaria species.     

Fatty acid composition of individual polar lipid classes from marine macrophytes

Major glycolipids [monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)) and phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG)] as well as betaine lipid 1,2-diacylglycero-O-4'-(N,N,N-tri-methyl)-homoserine (DGTS) were isolated from Anfeltia tobuchiensis (Rhodophyta), Laminaria japonica, Sargassum pallidum (Phaeophyta), Ulva fenestrata (Chlorophyta) and Zostera marina (Embriophyta), harvested in the Sea of Japan. GC analysis of their fatty acid (FA) composition revealed that the n-6 polyunsaturated FAs (PUFAs) shared the most part of the sum of n-6 and n-3 PUFAs in PC and PE compared with glycolipids and PG. In algae, it was related to the prevalence of 20:4n-6 over 20:5n-3 in non-photosynthetic lipids. Percentage of n-6 PUFAs as well as the sum of n-3 and n-6 PUFAs decreased in the following sequence: PC-->PE-->PG. The saturation increased in the lines of MGDG-->DGDG-->SQDG and PC-->PE-->PG. PG was close to SQDG by the level of saturation. Distribution of C(18) and C(20) PUFAs in polar lipids depended on taxonomic position of macrophytes. Balance between C(18) and C(20) PUFAs was preferably shifted to the side of C(20) PUFAs in PC and PE that was observed in contrast to glycolipids and PG from L. japonica containing both series of FAs. The set of major FAs of polar lipid classes can essentially differ from each other and from total lipids of macrophytes. For example, MGDG was found to accumulate characteristic fatty acids 16:4n-3, 16:3n-3, 18:3n-6 and 18:4n-3, 20:3n-6 in U. fenestrata, Z. marina, L. japonica and S. pallidum, respectively.