Nuclear-encoded protein-coding genes of the agarophyte Gracilaria verrucosa (Gracilariales,Rhodophyta)

Single-copy nuclear genes encoding cytosolic glyceraldehyde-3-phosphate dehydrogenase (GapC), triosephosphate isomerase (TPI1) and polyubiquitin (UBI6R), plastid-localized GAPDH (GapA), and mitochondrial aconitase (m-ACN), together with their corresponding cDNAs, have been cloned or PCR-amplified from Gracilaria verrucosa, sequenced, and characterized by genomic Southern hybridization. Three spliceosomal introns were found, one each near the 5 end of GapA, GapC, and m-ACN. Codon usage is biased, with third-position A's underrepresented. Nucleotide compositions of the genes are balanced except in TPI1. Upstream promoter structure, downstream poly(A) processing signals, intron splice junctions, and one translation initiation site were analyzed. Poly(A) processing features of these red algal genes resemble those of green plants more than those of animals.     

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.     

Highly methylated agar from Gracilaria edulis (Gracilariales, Rhodophyta)

The structure and gelling properties of alkali-modified agar from Gracilaria edulis were investigated. ¹H and ¹³C NMR experiments revealed a basic repeating unit of alternating 3-linked 6- O-methyl-β-D-galactopyranose and 4-linked 3,6-anhydro-α-L-galactopyranose. Partial methylation at O-2 of the anhydrogalactose moiety was also revealed. Meanwhile, the O-4 of the methylated galactose residue was detected to exhibit partial sulfation by NMR and FT-IR spectroscopy. The gel strength and syneresis index of the extracted agar were considerably enhanced by the addition of sodium, potassium, and calcium ions. The ion-driven gelation and peculiar sulfate position conferred the agar's similarity to κ-carrageenan. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Strain selection and genetic variation in Gracilaria chilensis (Gracilariales, Rhodophyta)

Strain selection processes in seaweed often have assumed that sterile clones could be maintained for long periods in a diversity of environments without major genetic changes. However, clonal species such as Gracilaria chilensis exhibit intra-clonal variation in performance and ongoing studies suggest such changes may be due to rapid changes in DNA composition associated with growth, via mitotic recombinations. Therefore performance of a given ramet in this type of seaweed should be understood as the dynamic outcome of rapid reactions between the environment and the changing genotype of the selected strain. To evaluate this idea, we measured changes in genetic variability, as detected by DNA-fragment polymorphism using RAPDs-PCR, exhibited by clones of G. chilensis after two transfers to different environmental conditions (from field to controlled laboratory conditions and from the laboratory to large-scale tank culture). The transfer to laboratory conditions reduced the frequency of low similarity values and increased the frequency of intermediate similarity values in DNA banding patterns, suggesting the branchlets produced under controlled laboratory conditions have less genetic variability (evaluated as total DNA polymorphism) than plants recently collected in the field. Tank incubation reduced the total range of similarity and significantly increased the frequency of high similarity values. Results thus suggest the dynamic of genetic changes in vegetative clones of Gracilaria chilensis that is fast and strongly affected by the external environment. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Integrated tank cultivation of salmonids and Gracilaria chilensis (Gracilariales,Rhodophyta)

In order to study the N and P balances in the Driss I reservoir, we measured concentrations in the water of these elements, their sedimentation rates, and their input by the river Inaouen and output through the dam. Supply and loss of N and P were calculated from samples collected every 48 hours and were for N 15 and 27. 5 mg m⁻² d⁻¹ and for P 33.6 and 1.1 mg m⁻² d⁻¹ respectively. The sedimentation rates, determined using sediment traps, were high, about 75 mg m⁻² d⁻¹ for N and 34 mg m⁻² d⁻¹ for P. Phosphate input came from point sources and was mainly in particulate form. A large fraction of the phosphate sedimented in the form of calcium bound phosphate, with some iron bound phosphate. Bioavailability was, however, low, as the particulate phosphate disappeared rapidly from the epilimnion because of the high sedimentation rate. The ratio Tot-N/Tot-P of the sedimented particulate matter varied between 0.05 and 7.74 and depended on primary production and watergate management.     

Studies on Gracilaria changii (Gracilariales,Rhodophyta) from Malaysian mangroves

Gracilaria changii, recorded from Malaysia and Thailand, is one of the more abundant agarophytic seaweeds found in Malaysia. A wild population of Gracilaria changii growing in mangroves was monitored for seasonal variation in agar content and gel strength as well as spore production. Agar yield and gel strength ranged from 12 to 25% dry weight and 294 to 563 g cm^–2, respectively, over a 15-month period. Gel strength but not yield was positively correlated with amount of rainfall. Cystocarps were observed throughout the study, but use of sporetraps showed that spore release peaked around July–August and January–February after the two monsoon periods.Mariculture of Gracilaria changii in shallow ponds in the mangroves, in an irrrigation canal and in a shrimp farm pond was conducted. Average growth rate of cuttings tied to monofilament lines was 3.3 ± 1.7% d^–1, 8.4 ± 1.8% d^–1 and 3.6 ± 1.6% d^–1 respectively. Cuttings were lost to siltation, wave action, predation and heavy epiphytisation.These studies show that Gracilaria changii has potential commercial application in the agar industry.     

Transient expression of lacZ in particle bombarded Gracilaria changii (Gracilariales, Rhodophyta)

Using a Biolistic PDS 1000/He system, healthy thalli of Gracilaria changii were bombarded with gold particles coated with plasmid DNA containing the lacZ reporter gene. Transient expression of lacZ was observed in bombarded thalli under the rupture-disc pressures of 4482, 6206, 7584 and 8963 KPa, two days after bombardment. Although G. changii exhibits a slight blue background, positive expression and the background colour can be clearly differentiated. The results indicate that lacZ could be a useful reporter gene and that SV40 promoter could be an effective promoter for Gracilaria transformation.     

Studies on the yield and gel strength of agar from Gracilaria domingensis Sonder ex Kuetzing (Gracilariales,Rhodophyta) following the addition of calcium

Studies were carried out on the seasonal variation in yield and gel strength of agar from Gacilaria domingensis with and without the addition of calcium chloride. Extraction was done with and without treatment with 1% hydrochloric acid. The results showed an increase in yield and gel strength when an alkaline solution of calcium was used, but the gel strength was low. For commercial use, Gracilaria domingensis should be mixed with better quality Gracilaria species because of its low gel strength.     

Experimental tank cultivation of Gracilaria sp. (Gracilariales,Rhodophyta) in Ecuador

This paper describes experiments to grow a local and still unidentified species of Gracilaria in shrimp hatcheries in Ecuador. The experiments used outdoor tanks of 1 and 18 m³ capacity, with continuous aeration and water renewal every two and five days, respectively. The sea water (salinity 34 ppt) was enriched with Guillard's f/2 medium; light and temperature were monitored but not controlled. One kg of fresh seaweed, inoculated into each tank, produced a biomass of ca. 3 kg in a period of 35 days in the 1 m3 tank and 18 kg in 43 days in the 18 m³ tank. We therefore believe that it is technically feasible to use the large infrastructure of existing shrimp hatcheries in Ecuador to produce Gracilaria.     

Cultivation of Gracilaria verrucosa (Gracilariales,Rhodophyta) Strain G-16 for agar

The agarophyte, Gracilaria verrucosa Strain G-16, has been grown in sustained cultivation over a period of five years. During this period, a number of experiments were conducted to examine the productivity, agar yield and agar gel strength of this strain. Productivities range from 3–31 g dry wt m^–2 d^–1 and are generally highest in the summer when annual water temperatures and daily irradiances are highest. In the summer months agar yield from Strain G-16 appears to be lowest whereas the gel strength of the agars was highest (> 750 g cm^–2).     

Stability of agar in the seaweed Gracilaria eucheumatoides (Gracilariales, Rhodophyta) during postharvest storage

The status of the cell-wall polysaccharide of the red seaweed, Gracilaria eucheumatoides upon postharvest storage was assessed in this study. The yield, chemical composition, physical and textural properties of alkali-treated agar extract was determined at different time intervals within 31 months of storage at dried state after harvest. Minimal fluctuation in agar yield was observed, ranging from 22.9% to 29.0%. The gel strength of agar extracts averaged 318gcm(-2) until the third month of storage but decreased considerably thereafter. The relative viscosity and molecular weight of the extracts varied inversely with storage time. Results indicated that both physical and textural parameters of agar generally decreased with storage time, likely due to depolymerization as indicated by decrease in molecular weight. Agar extracted from seaweeds up to 3 months of storage could be considered to exhibit gel quality suitable for food applications. Prolonged storage of the seaweed harvest is not recommended.     

Protoplast isolation optimization and regeneration of cell wall in Gracilaria gracilis (Gracilariales, Rhodophyta)

This paper reports the first successful isolation and cell wall regeneration of Gracilaria gracilis (Stackhouse) Steentoft, Irvine et Farnham protoplasts. These results form an important foundation for the development of a successful tissue culture system for G. gracilis. Initially, an isolation protocol was optimized by investigation of the effects of the enzyme constituents and concentrations, the pre-treatment of thalli, the incubation period and temperature, and the pH of the enzymatic medium on protoplast yields. A pre-treatment of G. gracilis thalli with 1 % (w/v) papain for 30 min followed by a 3-h enzymatic digestion of thalli with an enzymatic mixture containing 2 % (w/v) cellulase Onozuka R-10, 1 % (w/v) macerozyme R-10, and 10 U mL^?1 agarase at pH 6.15 was found to produce the highest yield of protoplasts at 22 °C. Reliably high yields (20–30?×?10⁵ protoplasts g^?1 f.wt) of protoplasts could be obtained from G. gracilis thalli when this optimized protocol was used. Cell wall re-synthesis by G. gracilis protoplasts, which constitutes the first step towards whole plant regeneration, was followed using calcoflour staining and scanning electron microscopy. Protoplasts were shown to complete the initial stages of cell wall re-synthesis within the first 24 h of culturing.     

Saldanha Bay,South Africa: recovery of Gracilaria verrucosa (Gracilariales,Rhodophyta)

Since World War II the greater Saldanha Bay lagoon system, South Africa, has been an important Gracilaria producer. Two agar factories, built in the 1960's, used Gracilaria from Saldanha Bay as their raw material. In the early 1970's the industry was destroyed as a result of dredging and marine construction operations to establish a harbor in the bay for loading ore. These environmental changes destroyed stocks and prevented the previously significant beachings of the seaweed from occurring. After a few years of no or very low commercial production, the resource slowly started to recover. The size of Gracilaria drifts increased over the following eight years to approximately one-third of the original output. This trend seems to continue. Although the stocks and resultant drifts are unlikely to recover fully to their original quantity, current production is already sufficient to ensure re-establishment of a seaweed industry in Saldanha Bay. This could have considerable socio-economic impact on the area.     

Characterization of an agar fraction extracted from Gracilaria dura (Gracilariales,Rhodophyta)

The evaluation of biopolymers of biotechnological interest has stressed the importance of renewable sources from the northern Adriatic Sea. In the frame of this research program, the red alga Gracilaria cf. dura, has been studied. Agar was obtained from Gracilaria cf. dura by means of alkali treatment and hot water extraction. In order to purify the extracts, amylase and isopropyl alcohol were used. Analysis of sugars was carried out by means of gas chromatography on alditol acetate derivatives; sulfate content was evaluated by means of infrared spectroscopy. An analysis of molecular weight distribution was carried out by means of high performance gel permeation chromatography coupled with a low angle laser light scattering device as detector. Analytical data were examined in terms of specificity of the algal source utilized, and the agar fractions extracted by means of this procedure were compared with a commercial sample.     

龙须菜免疫活性多糖的分离纯化及结构鉴定

利用小鼠淋巴细胞增殖模型,筛选龙须菜中具有免疫活性的多糖,并对其结构进行鉴定。活性筛选与分离纯化相结合,经室温提取,DEAE-Sepharose F.F.离子柱和凝胶Sephacryl S500层析,分离纯化得到具有刺激小鼠脾淋巴细胞增殖活性的龙须菜多糖GCpF2-3B。免疫实验表明,GCpF2-3B在较低浓度10μg/mL就表现出刺激作用(增殖率135%),当浓度为50μg/mL时增殖率达到最高(195%)。高效渗透色谱HPSEC层析纯度鉴定和紫外扫描表明GCpF2-3B为均一多糖,分子量约为1.03×105Da。红外光谱扫描显示,GCpF2-3B为典型的含硫酸基团多糖,具有糖类特征峰和总硫酸基吸收峰;13C NMR谱显示GCpF2-3B主要由交替相连的(1→3)-β-D-半乳糖和(1→4)-α-L-3,6-内醚半乳糖重复二糖组成。