Effects of UV-B radiation on growth rates,pigment content and ultrastructure of red (wild type), greenish-brown and green strains of Gracilaria birdiae (Gracilariales,Rhodophyta)

Based on physiological characteristics, we hypothesized that different strains of Gracilaria birdiae from two distinct geographical areas of the Brazilian coast (2500 km apart) would have different responses to long-term exposure to UV-B radiation (UV-B). The locations differ in their environmental conditions: one is a warmer area, Ceará State (CE), closer to the equator; the other is a colder area, Espirito Santo State (ES), closer to the Tropic of Capricorn. To test the hypothesis that the CE population is more resistant to UV-B than the ES population, apical segments of the red (RD_CE, RD_ES), green (GR_CE) and greenish-brown (GB_CE) strains were cultivated in the laboratory under two treatments: control (PAR) and artificial UV-B (PAR + UV-B). Algal performance was evaluated by considering growth rates, pigment content and ultrastructural analysis. Compared with the control, all strains showed a decrease in growth rates after exposure to UV-B. Of all strains, RD_ES showed the greatest sensitivity to UV-B. However, a decrease in growth rate and morphological changes were observed to a lesser extent in the RD_CE strain. Moreover, exposure to UV-B resulted in a decrease in the concentrations of phycobiliproteins in the RD_CE strain. The GB_CE strain showed an increase in phycoerythrin (PE)/allophycocyanin (APC) and phycocyanin (PC)/allophycocyanin (APC) ratios after exposure to UV-B, suggesting this strain had a higher tolerance to the radiation. No differences in the chlorophyll a and carotenoid content were found between the control and UV-B treated samples for all strains. Ultrastructural changes, such as damage to chloroplasts and mitochondria, were present in all strains after exposure to UV-B. In summary, our findings support the hypothesis that the population from Ceará State has adapted to the higher irradiation and is thus more resistant to increased UV-B. Additionally, of the strains tested, the GB_CE and RD_CE strains appear to be more resistant to this radiation.     

Color inheritance and pigment characterization of red (wild-type), greenish-brown, and green strains of Gracilaria birdiae (Gracilariales, Rhodophyta)

Species of Gracilaria are some of the most useful algae in the world for the production of agar. As a consequence of its economic importance, the genus has been the subject of many studies worldwide. Color variants of Gracilaria birdiae have been found in the natural population on the Brazilian coast, and they have also been isolated from plants cultivated in laboratory. These findings raised new questions regarding intraspecific variation and the prospects of cultivating such variants for their agar production. Therefore, this work aimed to determine the mode of color inheritance for two G. birdiae strains: a greenish-brown strain (gb) found in a natural population and a green strain (gr) which had arisen as a spontaneous mutation in a red plant cultured in the laboratory. The pigment contents of these strains, as well as the red wild-type (rd), were also characterized. Crosses between female and male plants of the same color (rd, gr, or gb) and between different colors were performed. Crosses between plants of the same color showed tetrasporophytic and gametophytic descendents of the parental color. Recessive nuclear inheritance was found in the greenish-brown strain, and cytoplasmic maternal inheritance was found in the green strain; both had lower phycoerythrin and higher concentrations of allophycocyanin and phycocyanin than the wild-type. Chlorophyll a contents were similar among all strains. Taken together, our results contribute to knowledge about the variability of this important red algae. In addition, since greenish-brown and green strains showed stability of color, both could be selected and tested in experimental sea cultivation to evaluate if mutants have advantageous performance when compared with red strain.     

Color inheritance, pigment characterization, and growth of a rare light green strain of Gracilaria birdiae (Gracilariales, Rhodophyta)

Gracilaria birdiae Plastino et E.C. Oliveira is an economically important marine red alga exploited for the production of agar in Brazil. A rare light green strain of G. birdiae was found in a natural population, which raised new questions regarding intraspecific variation. Crosses were performed in unialgal cultures to determine the mode of color inheritance of this light green strain. We determined the growth rate and pigment composition of the light green strain and compared it to the wild‐type, red strain. The light green color is stable and showed a recessive nuclear transmission. The light green strain had lower contents of chlorophyll‐a and phycobiliproteins (phycoerythrin, phycocyanin, and allophycocyanin), and grew more slowly than the red strain. This low performance is probably the reason why this mutant, although being stable, is so rare in nature. Nevertheless, it can be useful as a genetic visual marker and to investigate the structure and functioning of the photosynthetic apparatus.     

Intraspecific variation in Gracilaria caudata (Gracilariales,Rhodophyta): growth,pigment content,and photosynthesis

In Brazil, one of the probable reasons for failure in attempts at macroalgal mariculture is the lack of previous studies under controlled conditions. Gracilaria caudata is an important marine red alga which is locally exploited for the production of agar. In this study the aim was to compare in vitro growth rates, pigment content, and photosynthesis in gametophytes and tetrasporophytes of G. caudata from two distinct geographical areas located 2,500 km apart on the Brazilian coast, one in a warmer area closer to the equator (northeastern population), and the other in a colder area closer to the Tropic of Capricorn (southeastern population). Additionally, the artificial ultraviolet B (UVB) radiation effects on strains were evaluated. Under UVB, deleterious effects were observed in all strains. Although the strains from the southeastern population had higher growth rates than those from the northeastern under control condition, the opposite was observed under UVB condition. Under controlled conditions and regardless of the population, growth rates, net photosynthesis, P _max, I _k, and pigment contents were higher in tetrasporophytes than in gametophytes. Consequently, when determining the real potential of a certain phase in cultivation, the tetrasporophyte appears to be the more promising for future experiments along the Brazilian coast. Furthermore, although the growth rate of southeastern strains under control condition was higher, their higher sensitivity to UVB radiation emphasizes the importance of careful selection of the most suitable sites prior to experimental cultivation. The differences in performance between the southeastern and northeastern strains provide support for the hypothesis of their ecotypic differentiation.     

Polysaccharides from the red seaweed Gracilaria dura (Gracilariales, Rhodophyta)

The yield and physical and chemical properties of agars from Gracilaria dura (C. Agardh) J. Agardh, harvested in Thau lagoon (Mediterranean sea, France), were investigated. The agar yield ranged from 32% to 35%. Gel strength of agar ranged from 263 to 600 g cm(-2), with the maximum observed in October. A positive correlation was found between agar yield and gel strength (r = 0.82; P < 0.01). The gelling temperature followed the same pattern of gel strength and also showed higher value in October (43 degrees C). The nitrogen content varied from 1.04+/-0.60% (June) to 4.70+/-0.01% (October). A positive correlation was noted between nitrogen content and gel strength (r = 0.77; P < 0.05). The 3,6-anhydrogalactose content ranged from 0.70 to 0.84 and showed monthly significant differences (P < 0.05). There was a positive correlation between 3,6-anhydrogalactose content and gel strength. The values of sulfate content were relatively constant during the studied period and no significant differences were observed. The relative high gel strength indicates that this species may be considered as source of agar for commercial use.     

Growth,photosynthesis and agar in wild-type strains of Gracilaria verrucosa and G. conferta (Gracilariales,Rhodophyta), as a strain selection experiment

The local species Gracilaria conferta and the foreign G. verrucosa were grown together under a wide range of photon flux density and temperature conditions. Gracilaria verrucosa showed a higher growth rate, especially under low temperatures, and higher photosynthetic performances as well as higher ribulose-1,5-bisphosphate carboxylase activity as compared with G. conferta. Gracilaria verrucosa also showed a better quality and yield of agar, suggesting that this species could be more suitable than G. conferta for outdoor cultivation in Israel and may improve winter growth in ponds. Growth rate and agar quality (gel strength) were rated as the most suitable characteristics influencing the preference of strains for outdoor cultivation.     

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.     

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.     

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.     

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.     

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.     

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.     

Gracilaria corallicola and G. multipartita (Gracilariales,Rhodophyta), two related flattened European species

Two European species of Gracilaria possess flattened blades borne on cylindrical axes, namely, G. multipartita, known primarily from the Atlantic coast, and G. corallicola from the Mediterranean Sea. They are sister species that cluster with G. armata, G. bursa-pastoris and G. longa in rbcL analyses with strong bootstrap support. Blades of G. multipartita taper towards the tips, whereas those of G. corallicolla have broadly rounded tips. Spermatangia of G. corallicola are borne in shallow conceptacles (textorii-type) and data from the literature indicate that the same is true of G. multipartita. Cystocarp morphology is similar, with the gonimoblast filaments initially elongated, narrow and densely filled with cytoplasm, and with tubular nutritive cells issuing initially from lower gonimoblast cells and fusing with cells in the lowermost regions of the outer pericarp. Tetrasporangia are initiated terminally and displaced laterally with the production of side branches from the subterminal cell. The diagnostic characters of the Gracilariaceae are reviewed from a developmental perspective.     

Investigation of the Gracilaria gracilis (Gracilariales,Rhodophyta) proteome response to nitrogen limitation

Inorganic nitrogen has been identified as the major growth‐limiting nutritional factor affecting Gracilaria gracilis populations in South Africa. Although the physiological mechanisms implemented by G. gracilis for adaption to low nitrogen environments have been investigated, little is known about the molecular mechanisms of these adaptions. This study provides the first investigation of G. gracilis proteome changes in response to nitrogen limitation and subsequent recovery. A differential proteomics approach employing two‐dimensional gel electrophoresis and liquid chromatography–tandem mass spectrometry was used to investigate G. gracilis proteome changes in response to nitrogen limitation and recovery. The putative identity of 22 proteins that changed significantly (P < 0.05) in abundance in response to nitrogen limitation and recovery was determined. The identified proteins function in a range of biological processes including glycolysis, photosynthesis, ATP synthesis, galactose metabolism, protein‐refolding and biosynthesis, nitrogen metabolism and cytoskeleton remodeling. The identity of fructose 1,6 biphosphate (FBP) aldolase was confirmed by western blot analysis and the decreased abundance of FBP aldolase observed with two‐dimensional gel electrophoresis was validated by enzyme assays and western blots. The identification of key proteins and pathways involved in the G. gracilis nitrogen stress response provide a better understanding of G. gracilis proteome responses to varying degrees of nitrogen limitation and is the first step in the identification of biomarkers for monitoring the nitrogen status of cultivated G. gracilis populations.     

UVR defense mechanisms in eurytopic and invasive Gracilaria vermiculophylla (Gracilariales, Rhodophyta)

The invasive success of Gracilaria vermiculophylla has been attributed to its wide tolerance range to different abiotic factors, but its response to ultraviolet radiation (UVR) is yet to be investigated. In the laboratory, carpospores and vegetative thalli of an Atlantic population were exposed to different radiation treatments consisting of high PAR (photosynthetically active radiation) only (P), PAR+UV-A (PA) and PAR+UV-A+UV-B (PAB). Photosynthesis of carpospores was photoinhibited under different radiation treatments but photosystem II (PSII) function was restored after 12 h under dim white light. Growth of vegetative thalli was significantly higher under radiation supplemented with UVR. Decrease in chlorophyll a (Chl a) under daily continuous 16-h exposure to 300 μmol photons m(-2) s(-1) of PAR suggests preventive accumulation of excited chlorophyll molecules within the antennae to minimize the generation of dangerous reactive oxygen species. Moreover, an increase in total carotenoids and xanthophyll cycle pigments (i.e. violaxanthin, antheraxanthin and zeaxanthin) further suggests effective photoprotection under UVR. The presence of the ketocarotenoid β-cryptoxanthin also indicates protection against UVR and oxidative stress. The initial concentration of total mycosporine-like amino acids (MAAs) in freshly-released spores increased approximately four times after 8-h laboratory radiation treatments. On the other hand, initial specific MAAs in vegetative thalli changed in composition after 7-day exposure to laboratory radiation conditions without affecting the total concentration. The above responses suggest that G. vermiculophylla have multiple UVR defense mechanisms to cope with the dynamic variation in light quantity and quality encountered in its habitat. Beside being eurytopic, the UVR photoprotective mechanisms likely contribute to the current invasive success of the species in shallow lagoons and estuaries exposed to high solar radiation.     

Karyology and cytophotometric estimation of nuclear DNA content variation in Gracilaria,Gracilariopsis and Hydropuntia (Gracilariales,Rhodophyta)

Chromosome numbers of 1 N=24 were determined for three species of Gracilaria (G. flabelliforme P. Crouan et H. Grouan ex Schramm et Maze, G. mammillaris Montagne and G. tikvahiae McLachlan) and 1 N=32 for two species of Gracilariopsis (G. lemaneiformis (Bory) Dawson, Acleto et Folvik and G. tenuifrons (Bird et Oliveira) Fredericq et Hommersand). Karyotypes for these species exhibit a characteristic size difference between largest and smallest chromosomes. Polyvalents were a common feature of meiotic nuclei. Microspectrophotometry with the DNA-localising fluorochrome DAPI was used to quantify nuclear genome sizes. A 2 C genome size of 0·37–0·40 pg was determined for five species of Gracilaria (G. chilensis Bird, McLachlan et Oliveira, G. flabelliforme, G. mammillaris, G. pacifica Abbott, G. tikvahiae) and 0·33 pg for an isolate of G. verrucosa (Hudson) Papenfuss from Pas de Calais, France. Species of Hydropuntia (H. cornea (J. Agardh) Wynne and H. dentata (J. Agardh) Wynne) and Gracilariopsis (G. lemaneiformis and G. tenuifrons) were found to have slightly larger 2 C genome contents of 0·42–0·47 pg. No intraspecific variation in 2 C genome sizes was found in regional populations of Gracilaria tikvahiae and Gracilariopsis tenuifrons.     

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.     

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).