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.     

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.     

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.     

Content of arachidonic and eicosapentaenoic acids in polar lipids from Gracilaria (Gracilariales,Rhodophyta)

Fatty acid composition, especially the distribution of eicosapolyenoic acids in several species of Gracilaria, was analyzed in relation to their taxonomy. The species have been grouped into two types based on distribution of these polyenoic acids: Type 1, which contains palmitic, oleic and arachidonic acids as the major components, and Type II, which contains eicosapentaenoic acid in addition to Type I fatty acids. Octadecapolyenoic acids were detected only in trace amounts in each Type. A similar remarkable difference also was observed in the fatty acid composition of lipid classes. The major component of eicosapolyenoic acids in Type I was arachidonic acid in all lipid classes. In Type II, eicosapentaenoic acid was the major component in monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and phosphatidylglycerol. Arachidonic and eicosapentaenoic acids were contained in large amounts in Type II phosphatidylcholine. Grouping of Gracilaria species into Type I and Type II is not entirely consistent with morphological and taxonomic features, but the difference in fatty acid composition is likely due to genetic rather than to environmental factors.     

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.     

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.     

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).     

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.     

Evidence of reproductive cost in the triphasic life history of the red alga Gracilaria chilensis (Gracilariales,Rhodophyta)

The extent of changes in basic physiological and demographic traits associated with reproduction was investigated in the highly cultivated haploid–diploid red alga, Gracilaria chilensis. Sixty individuals bearing vegetative and reproductive fronds collected in the natural population of Niebla (39°52′?S, 73°23′?W), in Chile, were cultivated under controlled culture conditions. Our results demonstrated that vegetative fronds have a higher survival rate and a better growth rate than reproductive ones irrespective of the type of individual analyzed (male gametophyte, female gametophyte, and tetrasporophyte). Moreover, the reproductive fronds clearly showed a decrease in photosynthetic activity compared to non-reproductive ones. In males and tetrasporophytes, the photosynthetic reduction in reproductive individuals could be explained by a physical effect of reproductive structure development as well as spores release, disrupting the continuity of the photosynthetic cortical tissues. Translocation of photoassimilates from nearby vegetative tissue or the previous accumulation of photosynthetic products seems to be a prerequisite for reproductive structure development in this species. Altogether, these results document for the first time in G. chilensis that reproduction has a strong physiological effect on male, female, and tetrasporophyte fronds. This trade-off between reproduction, growth, and survival suggest the existence of reproductive costs in the life history of G. chilensis.     

Testing the effects of heterozygosity on growth rate plasticity in the seaweed Gracilaria chilensis (Rhodophyta)

Heterozygosity has been positively associated with fitness and population survival. However, the relationship between heterozygosity and adaptive phenotypic plasticity (i.e., plasticity which results in fitness homeostasis or improvement in changing environments) is unclear and has been poorly explored in seaweeds. In this study, we explored this relationship in the clonal red seaweed, Gracilaria chilensis by conducting three growth rate plasticity experiments under contrasting salinity conditions and by measuring heterozygosity with five microsatellite DNA markers. Firstly, we compared growth rate plasticity between the haploid and diploid phases. Secondly, we compared growth rate plasticity between diploids with different numbers of heterozygous loci. Finally, we compared growth rate plasticity between diploid plants from two populations that are expected to exhibit significant differences in heterozygosity. We found that, (i) diploids displayed a higher growth rate and lower growth rate plasticity than haploids, (ii) diploids with a higher number of heterozygous loci displayed lower growth rate plasticity than those exhibiting less heterozygosity, and (iii) diploid sporophytes from the population with higher heterozygosity displayed lower growth rate plasticity than those with lower heterozygosity. Accordingly, this study suggests that heterozygosity is inversely related to growth rate plasticity in G. chilensis. However, better genetic tools in seaweeds are required for a more definitive conclusion on the relationship between heterozygosity and phenotypic plasticity.     

Isolation and characterization of 19 polymorphic microsatellite DNA markers in the Japanese brown frog (Rana japonica)

We report the isolation and characterization of 19 polymorphic microsatellite DNA markers in the Japanese brown frog (Rana japonica). These markers were tested in 24 individuals each collected from three distinct populations in Ichikai-machi, Tochigi Prefecture. The number of observed alleles per locus ranged from 3 to 24 across all populations, and the values of observed and expected heterozygosities ranged from 0.130 to 1 and from 0.125 to 0.941, respectively. These microsatellite loci will be useful for investigating the intraspecific genetic variation and population structure of this species.     

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.     

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.     

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.     

Polymorphic microsatellite DNA markers for the Patagonian squid,Loligo gahi (Cephalopoda)

Squid populations are being put under rapidly increasing commercial fishing pressure worldwide. The same species are known to be susceptible to extreme population fluctuations, so detailed knowledge of population substructuring and genetic diversity is essential for rational management. We present a set of microsatellite DNA loci suitable for population genetic analysis of Loligo gahi, the squid species subject to the most detailed monitoring and fishery control (around the Falkland Islands), with the future aim of generating management‐related information to aid conservation of this valuable natural resource.     

Development of polymorphic microsatellite markers for the sorghum plant bug, Stenotus rubrovittatus (Heteroptera: Miridae)

Nine polymorphic microsatellite DNA markers from the sorghum plant bug, Stenotus rubrovittatus, were isolated and characterized. These markers were used to analyse 22 individuals from a single field population. The number of alleles at these nine loci ranged from two to 28 (mean = 11.4) and heterozygosity ranged from 0.27 to 0.86 (mean = 0.58). Stenotus rubrovittatus has shown rapid population growth in the decades since the first report in the 1980s of serious damage to a rice crop. These microsatellite markers will be of value for studying both the population genetics and population dynamics of S. rubrovittatus.     

Atlantic capelin (Mallotus villosus) tetranucleotide microsatellites

Twelve microsatellite loci developed for Atlantic capelin (Mallotus villosus) using magnetic bead hybridization enrichment for tetranucleotide microsatellites revealed five loci composed of single repeat elements and six composed of complex repeats. Forty‐four beach‐spawning females from three different northwestern Atlantic Newfoundland beach‐spawning populations were screened at each locus. Loci were polymorphic (two to 59 alleles per locus) and all but two exhibited high heterozygosity (0.86–1). The loci are considered suitable for addressing questions related to fine‐scale population structure, spawning fidelity and survivorship/kinship issues.     

Identification and characterization of microsatellites loci in Brycon opalinus (Cuvier, 1819) (Characiforme,Characidae, Bryconiae)

Brycon species are present in various basins in Brazil and were or still are part of commercial fisheries and aquaculture activities ( Ferreira et al. 1996 , Mendonça 1994 ). Despite the importance of this group of fish, natural populations of some Brycon species are endangered ( Lins et al. 1997 ). Here, we describe the characterization of seven microsatellite loci that will be useful for the genetic studies in natural and captive populations for these and other species of Brycon.