Temporal variations of macroalgal cover and biomass (wet weight and dry weight) abundance and assemblage structure, and also the relationships between abiotic factors and assemblage structure, were studied in northeastern Taiwan (GPS: 25o06¡¦80¡¨N; 121o54¡¦60¡¨E¡^during 2001-2003. This study also investigated the causes affecting Gelidium natural resource and its management. Twenty-seven families with 67 species (5 families with 13 species for Chlorophyta, 3 families with 8 species for Phaeophyta, 19 families with 46 species for Rhodophyta) were recorded. The dominant species such as Pterocladiella capillacea and Polyopes polyideoides belong to temperate species. Macroalgal coverage, areal biomass, diversity, eveness and assemblage structure during 2001-2003 showed significant temporal changes with year and seasonal variations; however, the seasonal rhythm is changing between years. The seasonal pattern is different between 2001 and 2002/2003, biomass peaked in spring of 2002 and 2003. Pterocladiella capillacea, Polyopes polyideoides, Marginisporum crassissimum, Jania ungulate and Chondrus ocellatus contribute to temporal variations in assemblage structure. The results of multi-variate and uni-variate analysis suggest significant temporal variations in macroalgal assemblage structure in northeastern Taiwan, and the data from ANOSIM and SIMPER analysis demonstrated that the seasonal pattern was variable between years, in which it is coincident with seasonal variations in macroalgal abundance. Water temperature, summer high temperature, winter low temperature, nutrient and typhoon are attributable to temporal variations in macroalgal assemblage structure. Low temperature in winter, nutrient availability, and also typhoon restrict the natural resource of Gelidium in northeastern Taiwan; it is hypothesized that low P condition together with enhanced collection deep the decline of Gelidium resource, finally limiting the recovery of Gelidium population in next year. We found that the agar contents of Pterocladiella capillacea showed year and season variations with a negative relationship to biomass. Temporal fluctuations in agar contents was enhanced by low temperatures and high P but decreased by high N. Based on the observations, we conclude that over-collection in winter results in a decline of Gelidium resource in the coming years, thus it could be suggested that winter collection has to be controlled to maintain the sustainable utilization of Gelidium in northeastern Taiwan.

Thesis (M.Sc. (Zoology & Entomology)) - Rhodes University, 2009

This dissertation reports a study of the fungi and 'protists' (Labyrinthulids, Thraustochytrids, Hyalochlorella marina) associated with the intertidal red algae Rhodoglossum affine and Gelidium coulteri. Research focused on laboratory isolations from algal thalli collected from in situ populations. Different isolation techniques and isolation media were employed to evaluate the abundance and diversity of fungi and protists associated with these red algae. Algal tissue surface sterilization and rigorous rinsing procedures were used to remove and/or enumerate surface-associated microbes. The results obtained from the different isolation techniques and algal tissue pretreatment procedures are compared and discussed in terms of their usefulness for each member of the algal-associated microbiota. Natural populations of affine and coulteri support a rich fauna of marine protists. The most prevalent members of this protist fauna were Labyrinthula spp. resembling the "Vishniac Strains" and Thraustochytrium motivum. Schizochytrium aggregatum, a new species of Labyrinthulid designated Labyrinthuloides sp. 1, and Hyalochlorella marina were also common depending upon the isolation method utilized. These protists appear to be associated with the surfaces of the algal thalli, and exist as saprobes and/or perthophytes rather than biotrophic parasites of the algal tissues. Isolations from field-collected algal tissues also yielded actinomycetes, yeasts, and a high diversity of imperfect fungi. Overall isolation frequencies for individual fungal taxa were low. Most of the mycelial fungi isolated are considered to be of terrestrial origin and of questionable 'significance' in the intertidal habitat. Only four, possibly five, are presently considered marine. The mycelial fungi most commonly isolated include: Acremonium sp. 019-78, Cladosporium cladosporioides, Dendryphiella salina, Penicillium spp., Phoma sp. (Group 1), Sigmoidea littoralis sp. nov., and Unidentified hyphomycete 044-78. Certain of these fungi may grow saprobically (as pertho-phytes) on reproductive and/or senescing algal tissues in the intertidal habitat, but their activities appear to be limited. Field-collected thalli of Rhodoglossum affine and Gelidium coulteri were allowed to decompose in mesh bags placed in the intertidal. The succession of higher fungi associated with the decomposing algae was followed by plating representative bimonthly subsamples of the algal tissues onto a Base Mineral Medium. Rhodoglossum affine deteriorated completely after 52 days of exposure, while a small amount of Gelidium coulteri remained after 71 days. Qualitative aspects of the mycobiota associated with the two algal species were similar, however fungi were isolated more frequently from coulteri. A dominant mycobiota was apparent after 36 days of exposure on the beach. Acremonium sp. 019-78, Dendryphiella salina and Sigmoidea littoralis sp. nov. were active colonizers of the decomposing algal tissues, and their isolation frequencies increased as decomposition proceeded. Several species of bacteria capable of utilizing the cell wall polysaccharides of red algae (agar, carrageenan) were also present on the decomposing algae. It is possible that the activities of these bacteria enhanced fungal development. Thraustochytrium motivum, Schizochytrium aggregatum and Ulkenia sp. RC02-80 were placed into sterile seawater cultures with surface-sterilized tissues of Rhodoglossum affine and Gelidium coulteri. After 72 hours of incubation, positive growth associations were examined using scanning electron microscopy. The three Thraustochytrids displayed luxuriant growth on all algal tissue types, and produced extensive ectoplasmic networks on the algal surfaces which functioned in attachment and, presumably, in the absorption of dissolved nutrients. Ectoplasmic net elements were resolved down to 0.02 pm in diameter, but no obvious 'penetration' of the algal tissues could be discerned. All of the protists (Labyrinthulids, Thraustochytrids, Hyalochlore11a marina) isolated from these red algae are described and illustrated. Certain commonly encountered and/or poorly known mycelial fungi are also described, including a new species of marine hyphomycete, Sigmoidea littoralis sp. nov. / Science, Faculty of / Botany, Department of / Graduate

L’objectif de ce travail est la valorisation des molécules bioactives présentes initialement dans Gelidium sesquipedale. Les rhodophycées agarophytes dont Gelidium sesquipedale sont exploitées à l’échelle industrielle pour l’agar, un phycocolloïde aux propriétés gélifiantes, qu’elles contiennent en abondance. Une multitude de coproduits sont générés lors de l’extraction de l’agar. Ces derniers, peu étudiés, ne sont pas valorisés alors qu’ils constituent une source potentielle de molécules d’intérêts. En premier lieu, le process industriel d’extraction de l’agar a été adapté à l’échelle du laboratoire afin de récupérer ces co-produits dont l’analyse a montré la richesse en glucides. Ils ont par la suite été fractionnés pour isoler les oligosaccharides dont certains sont connus comme éliciteurs chez les plantes. Ainsi, plusieurs fractions oligosaccharidiques ont été obtenues avec un rendement estimé à 15,7% de Gelidium sesquipedale sec. Les fractions sélectionnées ont été caractérisées par CPG, ESI-MS, RMN et perméthylation ce qui a permis d’élucider les structures des oligosaccharides qu’elles contiennent et de révéler notamment la présence de dérivés de floridoside dont le Gal2glycérol, le Gal3glycérol et le Gal4glycérol qui sont des molécules originales chez Gelidium sesquipedale non décrites à ce jour chez les algues rouges. Une dernière partie a consisté en la mesure de l’activité élicitrice de ces fractions qui a pu être vérifiée sur des plantes de tomate à travers des mesures de marqueurs biochimiques relatifs à l’expression des réactions de défense chez la plante. En conclusion, les coproduits issus de l’extraction de l’agar représentent une source de pSDNs (phyto stimulateur des défenses naturelles chez la plante) ; ils offrent une nouvelle perspective de développement à l’industrie de l’agar. / This work aims at promoting the bioactive molecules initially present in Gelidium sesquipedale. The rhodophycea agarophytes, including Gelidium sesquipedale, are used for industrial extraction of agar, a phycocolloid with gelling properties, which they contain in abundance. A multitude of co-products are generated during the extraction of the agar. These co-products have only been studied a little, hence not valued, while they constitute a significant source of molecules of interest. Firstly, the industrial agar extraction process was adapted on a laboratory scale, in order to recover these coproducts, which were subsequently subjected to an analysis, which revealed the presence of carbohydrates as major components. They were submitted to a fractionation process to obtain oligosaccharidic fractions, with a potential of elicitor activity, and a yield estimated at 15.7% of dry Gelidium sesquipedale. Also, a follow-up of co-products from batches of Gelidium sesquipedale harvested in different years from 2014 to 2016, enabled the comparison of the composition of the various co-products, depending on the year of the harvest, and thus to evaluate the variability of the initial resource. In addition, the impact of an extraction factor, being the sodium concentration, and the comparison with an industrial co-product produced by this process, were carried out. The retained fractions were characterized by GPC, ESI-MS, NMR and permethylation that allowed the elucidation of the structures of the oligosaccharides they contain, and revealed in particular the presence of floridoside derivatives including Gal2glycerol, Gal3glycerol and Gal4glycerol, which are original molecules in Gelidium sesquipedale, not described to date in red algae. A final part consisted in measuring the activity of these fractions as elicitor that could be estimated on tomato plants through measurements of biochemical markers relating to the expression of defense reactions in the plant. In conclusion, the co-products from agar extraction represent a source of pSDNs (phyto stimulator of natural defense in the plant) and give a new perspective to the agar industry.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Sulfated galactans (agarans and carrageenans) are accumulated in the cell wall of various red algae (Rhodophyta) species. These polysaccharides are of commercial importance in the food, pharmaceutical and biotechnology industries due to their unique physicochemical properties. Although having received significant research attention over the last 20 years, events regarding their biosynthesis have not been elucidated. Aiming for the identification of galactosyltransferase (GalT) genes involved in sulfated galactan biosynthesis, cDNA expression libraries were constructed from the prolific agar-producing South African red seaweed Gelidium pristoides (Turner) Kützing and screened by functional complementation of UDP-galactose 4-epimerase deficient mutants (E. coli and S. cerevisiae). Regretfully, no GalTs were identified. The study however yielded the first UGE enzyme described for a red seaweed. Southern hybridization indicated the presence of two UGE copies and confirmed the gene originated from G. pristoides. Bioinformatic analysis of G. pristoides UGE shows amino acid sequence homology to known UGEs from various organisms. The enzyme was shown to be functional in E. coli crude extracts and showed affinity for UDP-D-galactose, similar to other UDP-galactose 4-epimerases. Further, the isolated G. pristoides UGE (GpUGE) was biochemically characterized and its kinetic parameters determined. We found that there was no kinetic difference between this enzyme and previously described UGE enzymes except enhanced activity in the presence of exogenously added NAD+. The UDP-galactose 4-epimerase (UDP-glucose 4-epimerase, UGE, EC 5.1.3.2) is an essential Leloir pathway enzyme facilitating the catalytic inter-conversion between UDP-D-glucose and UDP-D-galactose. UDP-D-galactose is the nucleotide sugar required by galactosyltransferases for the production of red algae sulfated galactans. UGE is suspected as being responsible for supplying UDP-D-galactose for the synthesis of sulfated galactans. In planta monitoring of GpUGE transcript levels with respect to dark and light cycling indicated high expression of the enzyme at night, while expression diminished during the day. The occurrence of increased nocturnal UGE expression correlates with floridean starch breakdown at night. Evidence for hydrolysis of floridean starch is also reflected in obtained G. pristoides transcriptome sequence data. In red algae, floridean starch degradation coincides with sulfated galactan production. The detection of starch hydrolysis enzyme transcripts alongside increased expression of GpUGE suggests the enzyme plays a role in supplying UDP-Dgalactose for sulfated galactan production. As far as we know, this the first report of sequencing and biochemical characterization of a UGE from red seaweed.

Leucocin A is a bacteriocin produced by Leucoconostoc gelidium UAL 187-22. Bacteriocin producer strains possess an immunity protein, which enables the strain to protect itself against its own bacteriocin. The immunity gene from Leucoconostoc gelidium was isolated via PCR from a recombinant clone pJF5.5. This fragment was cloned by amplifying the immunity gene from pJF5.5 and ligating it into pMALc2. The resulting recombinant plasmid pKP1 was then transformed into Escherichia coli strain JM103. The clone putative, was confirmed by DNA sequencing and southern blot hybridization using the primers EAL-2 and EAL-3. It was shown to contain an insert of 3.6 kb. Expression analysis showed the construct as an in frame malE fusion protein expressed within E. coli. The fusion construct was isolated by affinity chromatography. Leucocin A was purified to 90% purity, from the supernatant of Leucocnostoc gelidium UAL 187-22 by ion-exchange chromatography and HPLC. It was found to elute from a C18 reverse phase column at 55% actetonitrile, 0.1% TFA. Binding interaction and the stability of the immunity gene fusion protein were compared using a Biacore 2000. The supernatant and cytoplasmic extract isolated from Leucocnostoc gelidium UAL 187-22 were tested for interaction with the fusion construct. Surface Plasmon resonance studies indicated that there was no binding partner present in the supernatant which would influence the immunity process. However, a stable interaction was found between the immunity protein and an orphan ligand within the cytoplasm. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

Southern African biogeographic boundaries delimit the phylogeographic distribution of some coastal and estuarine invertebrates. This study investigated the impact of these boundaries on the phylogeographic distribution of two intertidal red seaweeds, Gelidium pristoides and Hypnea spicifera using the mitochondrial Cox2-3 spacer and the nuclear ITS1 regions. G. pristoides spores have short distance-dispersal, while long distance-dispersal is more likely in H. spicifera via spores and drifting fertile thallus fragments. Both markers revealed a south-western and south-eastern lineage within G. pristoides but the breaks between lineages do not coincide with any recognised biogeographic limits. The Cox2-3 spacer revealed a boundary between the two lineages at the Alexandria Coastal Dunefield (ACD) and ITS1 at the Gamtoos-Van Stadens Dunefields (GVD) which is approximately 80km west of the ACD. The minor difference between the two markers regarding location of the phylogeographic boundary is probably due to the dating differences between the two dunefields. The ACD as developed currently is superimposed on the ancient dunefields which formed during the Pleistocene, coinciding with the Cox2-3 spacer sequences divergence which dates back 500,000 - 580,000 years. The GVD formed during the Holocene (6,500 - 4,000 years ago), coinciding with the ITS1 sequences divergence which dates 4,224 - 4,928 years ago. Thus, these phylogeographic boundaries probably appeared without the influence of biogeographic boundaries, but rather due to the lack of suitable habitat in the dunefields, coupled with short dispersal-distances of the spores. Analysis of the ITS1 and Cox2-3 spacer regions in H. spicifera revealed that the species is characterized by uniform genetic structure along the coastline. This reflects the species`s potential for long range expansion as it inhabits both the intertidal and subtidal zones; and this presumably leads to high gene flow among populations. The ITS1 sequences showed minimal genetic variation of one substitution between the gametophyte and tetrasporophyte generations within H. spicifera. This suggests the predominance of asexual reproduction, which reduces gene flow and fixes alleles between generations. ANOSIM and Bray-Curtis cluster analyses showed scale-dependant variation in the abundances of epifauna (mainly amphipod, isopod, mollusc and polychaete species) on G. pristoides. At small local (within site) and large (among sites) scales, there were weak and no structure in epifaunal abundances respectively. However, at larger, biogeographic scales, samples from the same biogeographic region tended to be clustered together. Thus, there was a group containing predominantly south coast samples and a group containing east coast samples mixed with the remaining south coast samples. Such scale-dependant variation in epifaunal abundances is probably due to the effects of factors driving species richness at small local (within site) scales (e.g. wave exposure, seaweed biomass) and at larger, biogeographic scales (e.g. surface sea temperature). Moreover, at very small (individual samples) scales; there was no correlation between epifauna composition and genotype of the seaweed. Seaweed samples characterized by distinct ITS1 or Cox2-3 spacer sequences did not show any significant differences in epifaunal composition. Although the distributional pattern of the epifaunal community observed at large biogeographic scale is not clear, it seems to be associated with the biogeographic regions. However, phylogeographic distribution of Gelidium pristoides is not connected to biogeographic regions. Thus, at larger, biogeographic scales, there is no correlation between phylogeographic distribution of G. pristoides and distribution of the associated fauna

The ecophysiology of the red alga Gelidium pristoides (Turner) Kuetzing was investigated in an effort to establish a technique for commercial cultivation. The seaweed is of commercial importance in South Africa where it is harvested from the intertidal zone rocky shores along the coast. It is dried and exported abroad for the extraction of agar. Yields and quality could be improved by cultivation in commercial systems. However, attempts at growing the seaweed in experimental systems have all ended in failure. This study aimed to describe the conditions in which the seaweed grows naturally; and investigate its physiological response to selected physical conditions in the laboratory in order to determine suitable conditions for mariculture. Ecological studies showed that G. pristoides grew above the spring low tide water level. The upper limit of the seaweed’s vertical distribution range, as well as its abundance, was largely dependent on wave exposure. The zone normally inhabited by G. pristoides was dominated by coralline turf in sheltered areas, while the abundance of G. pristoides increased towards more exposed rocky shore sites. The seaweed occurred among species such as Pattelid limpets and barnacles, but was usually the dominant macroalga in this zone, with coralline turf and encrusting algae being the only others. Physical conditions in the part of the intertidal zone inhabited by G. pristoides were highly variable. During low tide temperatures could vary by as much as 10°C within the three hours between tidal inundation of the seaweed population, while salinity varied by up to 9 ppt, and light intensity by as much as 800 μmol m-2 s-1. During these exposure periods the seaweed suffered up to 20% moisture loss. Laboratory experiments on the seaweed’s response to these conditions indicated that it was well adapted to such fluctuations. It had a broad salinity (20 and 40 ppt), and temperature tolerance range (18 to 24°C), with an o ptimum of temperature of 21°C for photosynthesis, while there was no difference in the photosynthetic rate of the alga within the 20 to 40 ppt salinity range. The alga had a low saturating irradiance (ca. 45 – 80 μmol m-2 s-1) equipping it well for photosynthesis in turbulent environments, with high light attenuation, but poorly to unattenuated light conditions. Exposure resulted in an initial increase in photosynthetic rate followed by a gradual decrease thereafter. pH drift experiments showed that low seawater pH, and associated increased carbon dioxide availability, resulted in an increase in photosynthetic rate. This response suggests that the seaweed has a high affinity for carbon dioxide, while the reduction in photosynthetic rate in response to bicarbonate use inhibition indicates that it also has the capacity for bicarbonate use. The high affinity of Gelidium pristoides for carbon dioxide as an inorganic carbon source appears to be the primary reason for the low abundance of the alga on sheltered rocky shore areas, and also explains the failure of the alga to grow in tank or open-water mariculture systems. Exposed rocky shores have experience heavy wave action, and the resultant aeration and mixing of nearshore waters increases the availability of carbon dioxide, which is considered a limiting resource. The absence of such mixing and aeration at sheltered site makes this less suitable habitat for G. pristoides. Periodic exposure also makes high levels of atmospheric carbon dioxide available from which the seaweed benefits. The traditional mariculture systems in which attempts have been made to cultivate the seaweed failed to satisfy either of the above conditions.