Gracilaria conferta and its epiphytes: 3. Allelopathic inhibition of the red seaweed byUlva cf.lactuca

In a previous study (Svirski et al., 1993), it was found that growth inhibition ofGracilaria spp., when cultured in the presence ofUlva cf.lactuca, was not due to shading or nutrient depletion, but seemed to be caused by competition for inorganic carbon or some type of allelopathy. In the present study, we attempted to differentiate between these two possible influences by (1) growing the two algae in biculture under various conditions, but keeping inorganic carbon levels constant and measuring net photosynthesis, respiration and growth rates, and by (2) measuring growth rates ofGracilaria spp. in the presence of extracts derived from media previously used to growUlva cf.lactuca.Both net photosynthesis and growth rates ofGracilaria spp. in biculture were inhibited, despite CO₂ (and also HCO₃ ^–) levels being kept constantly high in the culture media. It is likely that these responses were due to markedly enhanced rates of dark respiration inGracilaria spp. when grown together withUlva cf.lactuca. Growth ofGracilaria spp. was also inhibited by extracts derived from seawater in whichUlva cf.lactuca had previously been grown. The strong inhibition by ethyl acetate and chloroform extracts indicate an allelopathic effect onGracilaria spp.     

Gracilaria conferta and its epiphytes: (2) Interrelationship between the red seaweed and Ulva cf. lactuca

Ulva cf. lactuca has been a disturbing competitor of experimental Gracilaria conferta outdoor cultures in Israel. The effect of environmental conditions on the competitive ability of Ulva versus Gracilaria, and the main limiting factors for which these seaweeds compete, were studied here. Single and biculture experiments of both seaweeds showed that Ulva outgrew and damaged Gracilaria under all irradiance and temperature combinations. The higher competitive ability of Ulva cf. lactuca in bicultures was not a result of responses to shading or nitrogen shortage, but rather to a shortage of available inorganic carbon, an increase in pH and apparent excretions of Ulva which inhibited the growth of Gracilaria.     

Associational plant refuges: convergent patterns in marine and terrestrial communities result from differing mechanisms

Summary An associational plant refuge occurs when a plant that is susceptible to herbivory gains protection from herbivory when it is associated with another plant. In coastal North Carolina, the abundance of the palatable red alga Gracilaria tikvahiae is positively correlated with the abundance of the unpalatable brown alga Sargassum filipendula during times of increased herbivore activity. To see if grazing by the sea urchin Arbacia punctulata could generate this pattern, controlled experiments were conducted in out-door microcosms and in the laboratory. Gracilaria beneath a canopy of Sargassum was eaten significantly less than Gracilaria alone. When Arbacia were excluded, Gracilaria alone grew significantly more than Gracilaria beneath Sargassum, demonstrating that Sargassum is a competitor of Gracilaria. Experiments investigating Sargassum's deterrent role indicated that Sargassum decreased the foraging range of Arbacia and the rate at which it fed on Gracilaria. Additional experiments with plastic Sargassum mimics indicated that the decreased grazing on Gracilaria was not a result of Sargassum morphology, but was probably attributable to some chemical characteristic of Sargassum. The pattern of increased grazing in monocultures (only Gracilaria present) versus polycultures (both Gracilaria and Sargassum present) demonstrated in this study also has been demonstrated for plant-insect interactions in terrestrial communities. In these communities, insect density is higher in monocultures than in polycultures because insects find and immigrate to monocultures more rapidly, and once in a monoculture, they emigrate from them less often than from polycultures. In this study, urchins did not find and immigrate to monocultures more rapidly, nor did they tend to stay in them once they were found; in fact, they emigrated from monocultures of Gracilaria more rapidly than from Gracilaria and Sargassum polycultures. Increased grazing in Gracilaria monocultures resulted from increased rates of movement and feeding of individual herbivores, not from increased herbivore density as has been reported for terrestrial systems.     

DEFENSE EVOLUTION IN THE GRACILARIACEAE (RHODOPHYTA): SUBSTRATE‐REGULATED OXIDATION OF AGAR OLIGOSACCHARIDES IS MORE ANCIENT THAN THE OLIGOAGAR‐ACTIVATED OXIDATIVE BURST1

Combined phylogenetic, physiological, and biochemical approaches revealed that differences in defense‐related responses among 17 species belonging to the Gracilariaceae were consistent with their evolutionary history. An oxidative burst response resulting from activation of NADPH oxidase was always observed in two of the subgenera of Gracilaria sensu lato (Gracilaria, Hydropuntia), but not in Gracilariopsis and in species related to Gracilaria chilensis (“chilensis” clade). On the other hand, all species examined except Gracilaria tenuistipitata var. liui and Gracilariopsis longissima responded with up‐regulation of agar oligosaccharide oxidase to an challenge with agar oligosaccharides. As indicated by pharmacological experiments conducted with Gracilaria chilensis and Gracilaria sp. “dura,” the up‐regulation of agar oligosaccharide oxidase involved an NAD(P)H‐dependent signaling pathway, but not kinase activity. By contrast, the activation of NADPH oxidase requires protein phosphorylation. Both responses are therefore independent, and the agar oligosaccharide‐activated oxidative burst evolved after the capacity to oxidize agar oligosaccharide, probably providing additional defensive capacity to the most recently differentiated clades of Gracilariaceae. As demonstrated with Gracilaria gracilis, Gracilaria dura, and Gracilariopsis longissima, the different responses to agar oligosaccharides allow for a fast and nondestructive distinction among different clades of gracilarioids that are morphologically convergent. Based upon sequences of the chloroplast‐encoded rbcL gene, this study suggests that at least some of the samples from NW America recorded as Gs. lemanaeiformis are probably Gs. chorda. Moreover, previous records of Gracilaria conferta from Israel are shown to be based upon misidentification of Gracilaria sp. “dura,” a species that belongs to the Hydropuntia subgenus.     

World-wide use and importance of Gracilaria

The world's first source of agar, from the middle of the seventeenth century, was Gelidium from Japan, but by the beginning of the twentieth century demand for the phycocolloid exceeded of the supply of this alga. Since then Gracilaria has played an important role in the production of agar. Currently agars are obtained from five genera in three orders of red algae and marketed as ‘natural agar’ in squares or strips or as ‘industrial agar’ in powder form. The development of production processes through alkaline hydrolysis of sulphates has allowed a good quality food agar to be obtained from Gracilaria. This does not show the synergistic reaction with locust bean gum apparent with Gelidium agar. The term ‘agaroids’ is applied to Gracilaria agars produced without alkaline hydrolysis of sulphates, with greater sulphate content and much less gel strength. Unlike Gelidium, Gracilaria has to be processed in a short period of time and cannot be allowed to remain in storage for use during years of lower availability. Statistics of imports of agarophytes to Japan during the last 10 years give an indication of the state of the market. During this period there was a marked reduction in Gracilaria imports, mainly from Chile, but also the Philippines, Indonesia and South Africa, mainly due to the overall increase in the capacity of agar production in Gracilaria-producing countries.     

Using gigartinine to track the distribution of an alien species of <Emphasis Type="Italic">Gracilaria</Emphasis> in New Zealand

The presence of gigartinine has been used previously as a taxonomic marker for the positive identification in Manukau Harbour (west coast, Auckland) of Gracilaria sp., which has apparently been introduced to New Zealand waters and is easily confused morphologically with the native species, G. chilensis. Thirty two estuarine/harbour populations of Gracilaria in New Zealand were screened for the presence of gigartinine to further test the utility of gigartinine as a reliable species marker. DNA fingerprinting was used to confirm the identity of a subset of the specimens surveyed. Using genetic rather than chemical characterisation, it was discovered that Gracilaria sp. is also present in Orakei Basin (east coast, Auckland). Although a sample from the wild did not have the anticipated gigartinine content, tank cultivation of thalli from this population in an artificially elevated nitrogen environment allowed the plant to accumulate nitrogen as gigartinine. This confirmed the unusual ability of this species of Gracilaria to store nitrogen in this form, unlike the widespread, morphologically similar, G. chilensis.     

Cultivation of Gracilaria on the sea-bottom in southern Chile: a review

This review contains information about the cultivation techniques, strategies, problems and new challenges faced as well as an economic analysis of the income-producing capacity of Gracilaria farming, considering the variability of environmental systems where this alga is cultivated in southern Chile. The development of Gracilaria farming in Chile was made possible by an increased market demand, as well as the existence of basic knowledge that permitted the management of wild stocks and the initiation of cultivation practices. Subtidal cultivation systems appear to be more productive than intertidal systems and are less susceptible to wave action than intertidal cultivation areas. In relation to farming practices, this difference implies that planting and harvesting methods and strategies vary between habitats where cultivation is being carried out on a commercial scale. Several problems such as the environmental impact of different cultivation methods adopted by the farmers, the management of contaminating organisms and strain selection appear to be important and new areas for future research. Finally, an analysis of the income-producing capacity indicates that environmental differences also have important consequences for the management strategies of Gracilaria cultivation.     

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.     

Recent trends of seaweed production in Chile

In the last fourteen years the production of seaweeds in Chile has ranged from 74 000 to 229 000 wet metric tons per year and has included about twenty species belonging to Phaeophyta and Rhodophyta. The only source of this production has been the exploitation of natural beds, except for Gracilaria, which is the only case of commercial cultivation and contributes significant quantities to total production. Initially most of the raw material was exported but currently important quantities of Gracilaria and several carrageenophytes are being processed by local industry. Changes in production of the main resources are analyzed with consideration of potential demand, level of knowledge about natural beds, and the situation of total Gracilaria farming, in order to attempt predictions for the supply. Current possibilities of applying new technologies to cultivate other economically important Chilean seaweeds are also analyzed and discussed.     

The effect of localised eutrophication on competition between Ulva lactuca (Ulvaceae,Chlorophyta) and a commercial resource of Gracilaria verrucosa (Gracilariaceae,Rhodophyta)

In spring (August) 1993 a bloom of Ulva lactuca appeared for the first time in Saldanha Bay, South Africa, and persisted through summer. Ulva wash-ups contaminated the beach and part of the commercial Gracilaria beach-cast had to be discarded. The biomass and distribution of Gracilaria and Ulva are described in relation to the seasonal water chemistry of the bay. Gracilaria survives in deeper water in summer by the pulsing of nutrients on an approximately 6-day cycle of movement of the thermocline that separates nutrient-rich bottom water from warm oligotrophic surface water. Ammonium-rich fish-factory discharge into this surface layer in a sector of the bay provided localised conditions for Ulva to out-compete Gracilaria at depths of 2–5m, demonstrating the powerful disruptive effect of eutrophication in this strongly stratified system.     

Recent status of Gracilaria cultivation in Taiwan

The recent decrease in Gracilaria culture production and value in Taiwan were evaluated from statistical data and from interviews with local fishermen. Reasons are: 1) during 1986–87, many Gracilaria culture ponds were transformed to grow grass shrimp (Penaeus monodon) in monoculture, but disease of the shrimp occurring soon after stopped such production and Gracilaria culture took over, but 2) due to manpower shortage, Gracilaria-farmers prefer to sell their crops to abalone farmers and not to agar factories. Since Gracilaria as abalone feed is cheeper than for agar production, the value of algal crop decreased.     

Opportunities and challenges for the development of an integrated seaweed-based aquaculture activity in Chile: determining the physiological capabilities of <Emphasis Type="Italic">Macrocystis</Emphasis> and <Emphasis Type="Italic">Gracilaria</Emphasis> as biofilters

Seaweed production is a reality in Chile. More than ten species are commercially used to produce phycocolloids, fertilizers, plant growth control products, human food or animal fodder and feed additives. These multiple uses of algae offer a number of possibilities for coupling this activity to salmon, abalone and filter-feeder farming. In this context, different experiments carried out in Chile have demonstrated that Gracilaria chilensis and Macrocystis pyrifera have great potential in the development of an integrated aquaculture strategy. The present Integrated Multi-Trophic Aquaculture (IMTA) approach study showed that Gracilaria can be cultured best at 1 m depth whereas Macrocystis has an especially good growth response at 3 m depth. Both species use available nitrogen efficiently. On the other hand, high intensities of solar radiation (UV and PAR) can be critical at low depths of cultivation, and our results indicate that both species show photosynthetic susceptibility mainly at noon during the summer. The demand of Macrocystis for abalone feeding is increasing, thus improving the opportunity for developing an integrated nutrient waste recycling activity in Chile. Although Gracilaria shows a higher nitrogen uptake capacity than Macrocystis, its market value does not yet allow a massive commercial scaling.     

Application of <Emphasis Type="Italic">Gracilaria lichenoides</Emphasis> (Rhodophyta) for alleviating excess nutrients in aquaculture

A study was conducted in Xiangshan Bay, Ningbo, China, using red alga Gracilaria lichenoides to alleviate nutrient pollution in shrimp (Litopenaeus vannamei) and fish (Epinephelus awoara) culture ponds. Our results showed that G. lichenoides was efficient at absorbing inorganic nitrogen (IN) and inorganic phosphate (IP), and maintained a more stable dissolved oxygen (DO) level. A total of 506.5 kg (1,013 kg ha⁻¹) of shrimp and 210.5 kg (421 kg ha⁻¹) of fish were harvested from the shrimp/algae (SA) and fish/algae (FA) ponds, respectively. Only 53.5 kg shrimp were harvested from the shrimp pond without Gracilaria (S) due to anoxic asphyxia, and 163 kg fish were harvested from the fish culture pond without Gracilaria (F). Compared with using microalgae, bioremediation by macroalgae has no risk of harmful algal blooms (HABs), and it is easy to control seaweed biomass. During the experiment, there was a better environmental condition (lower chemical oxygen demand, IN, IP and chlorophyll a concentrations) in the ponds with Gracilaria. Furthermore, Gracilaria spp. can be used as food for abalone or other aquacultured animals and thus enhance economic return.     

The presence of gigartinine in a New Zealand Gracilaria species

Gigartinine, 5-(3-amidinoureido)-2-aminovaleric acid, serves as achemotaxonomic marker to distinguish two species of Gracilaria withvery similar morphologies. Gigartinine was identified by ¹³C-NMRspectroscopy and amino acid analysis of a cold-water extract from Gracilaria sp. nov., collected from a sheltered harbour localityat Blockhouse Bay, Auckland, New Zealand. Levels of this amino acid,naturally ca. 5% by dry weight of seaweed, were able to be depleted andthen restored during a nitrogen pulsing experiment. In contrast, native andpulsed samples of Gracilaria chilensis from Point Arthur, Wellingtonshowed no extractable gigartinine. Although these two species are unableto be distinguished in the field by morphological characteristics, they canbe separated by the presence or absence of gigartinine.     

Chemical structure and quality of agars from Gracilaria

Agar polymers synthesized by species of the genus Gracilaria constitute a complex mixture of molecules, containing several extremes in structure. Sulphate hemi-esters, methyl ethers and pyruvic ketals can alter in a number of ways the structural regularity of agar based on strictly 3-O-linked β-l-galactopyranose and 4-O-linked α-l-galactopyranose residues. In comparison with agars from Gelidium and Pterocladia, agars from Gracilaria can have higher degrees of sulphation, methoxylation and pyruvylation. The gelling ability of agars from most of Gracilaria species is considerably improved by adopting, before extraction, an alkali pretreatment which converts α-l-galactose 6 sulphate into 3,6-anhydro-α-l-galactose. Native agars obtained from Gracilaria cannot be classified, with few exceptions, as bacteriological grade agar as they have a high content of methoxyls and consequently high gelling temperatures. On the contrary, the genus Gracilaria is considered the most important source of food and sugar-reactive grade agars. Among techniques which can be used to study algal polysaccharides, combined ¹H and ¹³C nuclear magnetic resonance spectroscopy represent the most effective and powerful method for the investigation of the chemical structure of agarocolloids.     

Assessment of populations ofGracilaria chilensis (Graeilariales,Rhodophyta) utilizing RAPDs

Phenotypic variability and mixing of material due to massive cultivation for commercial purposes has contributed to the taxonomic confusion ofGracilaria in Chile. At least four species with cylindrical thalli and similar morphology have been recorded. However, since establishment ofG. chilensis, most of the collected thalli have been attributed to this species despite the lack of diagnostic features. In an attempt to resolve whetherGracilaria from 3 localities where it grows in natural and artificial populations belongs to the same species, gametophytic samples were compared by applying RAPD-PCR to their total DNA. This was analysed using 25 different 10-mer primers from which 21 revealed polymorphism within and between populations. Similarity matrices and cluster analyses were performed based on the presence/absence of bands representing fragments of DNA generated by random amplification. Similarity values between two of the populations were equivalent to those detected within a third, indicating the mixing of genetic material due to transplant between the two former localities. Similarities between samples of ChileanGracilaria andG. tenuistipitata from Sweden are considerably lower (0.45–0.53) than those between populations from Chile (0.74–0.88), confirming the existence of a single specific taxon,G. chilensis, in these three localities.     

Agar yield and quality of Gracilaria chilensis (Gigartinales,Rhodophyta) in tank culture using fish effluents

Tank cultivation of Gracilaria using fish effluents has permitted a production of 48 kg m^–2 yr^–1 and can reduce the dissolved nitrogen loads in the seawater. We report the yield, gel strength, gelling and melting point of agar from Gracilaria cultivated in tanks with seawater previously utilized in intensive, land-based salmon cultures and compared to a control using directly pumped seawater, over a study period of 22 months. The results show that the highest agar yield (20 to 22%) was obtained when Gracilaria was cultivated with pure seawater as compared to the fish effluents. The gel strength, gelling and melting point were higher in the agar obtained from algae cultured with fish effluents. During the spring, the gel strength, gelling and melting point increased in tanks with fish effluents and decreased in tanks with a supply of pure seawater.     

Gracilaria and its epiphytes: 4. The response of two Gracilaria species to Ulva lactuca in a bacteria-limited environment

The responses of Gracilaria lemaneiformis, an easily epiphytized host,and the relatively resistant G. cornea mutant, to the green alga Ulva lactuca were studied using biculture experiments with and withoutantibiotics. Both Gracilaria species grown with and without U.lactuca showed different levels of growth rate, release of hydrogenperoxide and of halogenated hydrocarbons. These quantitative differencesled to a successful response against Ulva lactuca in the case of G.cornea mutant and to a failure in response in the case of G.lemaneiformis. The response of each Gracilaria species to U.lactuca was qualitatively similar to its response to bacteria. This suggeststhe involvement of oligosaccharide elicitors produced in the presence ofepiphytes and bacteria. A clear Gracilaria inhibition was demonstratedwith extracts of the culture medium. It appears that hydrogen peroxide,halogenated hydrocarbons and oligosaccharides may be components of theinhibitory activity of the extracts. The responses of Gracilaria speciesto the presence of U. lactuca suggest the characterization of adefence response.     

Effects of marine algal proximate composition on methane yields

The suitability of differentGracilaria spp. and twoSargassum species for bioconversion to methane was determined through bioassays of methane yield.Gracilaria species and strains were excellent feedstocks for high methane yields, ranging from 0.28 to 0.40 m³ kg^–1 volatile solids added. These yields ranged from 58 to 95% of theoretical stoichiometric yields. Methane yields were highly correlated with acid soluble carbohydrate components of theGracilaria spp. BothSargassum fluitans andS. pteropleuron were poor feedstocks, with methane yields ranging from 0.12 to 0.19 m³ kg^–1 volatile solids added, 27 to 46% of theoretical stoichiometric yields, respectively. The various tissue types of theseSargassum species were also poor feedstocks for anaerobic digestion to methane. While there is no clear explanation for the low methane yields, the twoSargassum spp. appear to contain a high proportion of an insoluble, non-extractable component which may not be available as a substrate for bioconversion to methane.     

Intercellular assimilate translocation in Gracilaria cornea (Gracilariaceae,Rhodophyta)

Assimilate translocation has been identified and characterized in Gracilaria cornea under different conditions. Carbon fixation and translocation were carried out by inserting the base part of the thallus into a bicarbonate labeled solution in seawater and exposing its upper part to the air (open system) or to a non-labeled solution above a rubber septum (closed system). After a pulse-chase treatment in the light, three separate sections of each thallus were extracted by DMF (high moleuclar weight photosynthates) or by ethanol (low molecular weight). The results indicate a high rate of active photosynthate translocation which is directly related to inorganic carbon gradients in the thallus, and probably also to sugar gradients in the thallus. Translocation parameters of Gracilaria cornea are lower than of brown algae, as Gracilaria does not contain specific translocation tissues.