Relationships among Eucheuma denticulatum,Eucheuma isiforme and Kappaphycus alvarezii (Gigartinales,Rhodophyta) based on nuclear ssu-rRNA gene sequences

The nuclear genes encoding small-subunit ribosomal RNAs (ssu-rDNAs) of the carrageenophytes Eucheuma denticulatum, E. isiforme and Kappaphycus alvarezii were amplified by the polymerase chain reaction, cloned and sequenced. The sequences range from 1767 (K. alvarezii) to 1781 (E. isiforme) nucleotides in length, and have guanine+cytosine contents between 51.2% (E. isiforme) and 51.5% (E. denticulatum). Pairwise sequence identities among these sequences ranged from 97.6% to 98.5%, levels comparable to some intergeneric identities within Gracilariales. In phylogenetic analyses, the two Eucheuma ssu-rDNAs group stably together vis-a-vis the ssu-rDNA of K. alvarezii, and these three ssu-rDNAs form a monophyletic group within a larger grouping of other carrageenophytes. The results demonstrate quantitatively that analysis of nuclear-encoded ssu-rDNA sequences is likely to be useful in resolving taxonomic, phylogenetic and biogeographic questions among tribe Eucheumatoideae Doty.     

‘Endong’, a rare variety of the farmed carrageenophyte Eucheuma denticulatum (Burman) Collins & Hervey from the Philippines

The variety described in this paper is rather a rare and interesting one. Locally called ‘endong’, this was discovered first in a farm in Tawi-Tawi, Sulu Archipelago, Philippines. During harvest, seaweed farmers used to mix this with ‘tambalang’ (Kappaphycus alvarezii (Doty) Doty ex Silva var. tambalang Doty, the ‘cottonii’ of commerce) because of the presumption of it producing the same carrageenan type as that of ‘tambalang’, viz., kappa-carrageenan. The two farmed varieties are similar in appearance. Complaints arose from seaweed processing plants when the resulting gelling properties (hard and brittle gel) expected of the kappa-carrageenan-producing ‘tambalang’ were not met. Because of its lax habit and long, smooth, and naked basal axes, ‘endong’ was initially thought to be related to ‘tambalang’ and differ from the farmed ‘spinosum’ (Eucheuma denticulatum (Burman) Collins & Hervey) whose typical or common form has shorter axes covered from base to apex with spinous determinate (ramuli) or indeterminate branchlets arranged irregularly and in whorls at predictable interval. However, a closer look at ‘endong’ shows that it also has spinous branchlets arranged in whorls, but confined to the apical portion of the branches. Moreover, its internal axial anatomy shows rhizoidal filaments forming a core. Both characters fit well the distinguishing taxonomic criteria established for E. denticulatum. Chemical characterization of its carrageenan and the determination of its phylogenetic affinities support our morphological findings. Hence, we report and describe ‘endong’ as a rare variety, and we name it E. denticulatum (Burman) Collins & Hervey var. endong Trono & Ganzon-Fortes var. nov. Tips to distinguish ‘endong’ from ‘tambalang’ are included in this paper to aid seaweed farmers in separating the two during harvest.     

Karyology,nuclear genome quantification and characterization of the carrageenophytesEucheuma andKappaphycus (Gigartinales)

DNA reassociation kinetics were used to determine nuclear genome organization and complexity in the carrageenophyteKappaphycus alvarezii. Results indicate the presence of three second order components corresponding to fast (12%), intermediate (38%) and slow (50%) fractions. Microspectrophotometry with the DNA-localizing fluorochrome DAPI confirmed ploidy level differences in the gametophytic and tetrasporophytic phases. Comparison of mean nuclear DNA (I _f ) values to chicken erythrocytes (RBC) resulted in pg/2C genome estimates:Eucheuma denticulatum=0.35,E. isiforme=0.44,Kappaphycus alvarezii=0.32 andK. striatum=0.42. Karyological studies of tetraspore mother cells during diakinesis using aceto-orcein revealed a chromosome complement of 10 forEucheuma denticulatum andKappaphycus alvarezii.     

Inhibitory activities of three Malaysian edible seaweeds on lipase and α-amylase

Ethanol extracts, dried powders and fibres (total and soluble fibre) of the tropical red algae Kappaphycus alvarezii, Kappaphycus striatus and Eucheuma denticulatum were analysed for their effect on lipase and α-amylase activity using turbidimetric method and dinitrosalicylic acid (DNS) assay, respectively. The nutrient composition analyses were determined using standard methods. The ethanol extract of dried K. striatus (Ks-III) showed the highest reduction in lipase activity with 92 % inhibition followed by seaweed powders (K. alvarezii (Ka-III), K. striatus (Ks-III) and E. denticulatum (Ed-III)) with average inhibition of 60 %. Soluble fibres of K. alvarezii (Ka-V) and E. denticulatum (Ed-V) showed significant inhibition with 60 and 57 % reduction, respectively. Only the ethanol extract of fresh E. denticulatum (Ed-I) showed 88 % inhibition of α-amylase. Nutritional component analyses showed that all three seaweeds are low in crude fat, suggesting the possible use of seaweed as a dietary supplement and for potential weight and glycaemia management.     

Aspects of the farming and processing of Kappaphycus and Eucheuma in Indonesia

Indonesia has become the largest exporter of Eucheuma denticulatum, supplying the foreign demand for iota-carrageenan, and the increasing demand of the Asian food markets. Recent demand fluctuations for Kappaphycus alvarezii and E. denticulatum have resulted in price instability, and slowed the expansion of the less viable raft culture practice. A short 42–45 day harvest interval, propagule selection, and post-harvest treatment remain the causes of poor crop quality and low carrageenan yields.In 1988 the first processing of K. alvarezii commenced. By 1991 there were four factories exporting an alkali-treated chip product, and one producing carrageenan powder. In 1991, 3200t dry weight of the K. alvarezii harvest was processed locally. The viability of future natural food-grade carrageenan production is discussed in relation to new developments in the industry.     

Temperature acclimation in culturedEucheuma isiforme from Florida andE. alvarezii from the Philippines

Branch cultures ofEucheuma alvarezii Doty var.tambalang Doty, farmed in the Philippines, andE. isiforme (C. Agardh) J. Agardh var.denudatum Cheney, from the west coast of Florida, were gradually transferred through three temperature regimes over a 6-week period. Photosynthetic responses were measured under a series of irradiances (P-I curves) and temperatures to determine immediate responses of the plants before, during and after completion of the transfers. The Philippine variety did not show acclimation to 18 °C either after gradual transfer from the initial culture temperature of 25 °C or when abruptly transferred from 25 to 18 °C. The Florida variety did show acclimation to 25 °C when gradually transferred from 18 to 22 to 25 °C over the 6-week period, but not if abruptly transferred from 18 to 25 °C. The west coast variety ofE. isiforme from Florida shows a temperature acclimation ability that parallels the seasonal changes in water temperature of its habitat.     

Gelling interactions of phycocolloids extracted from red algae with a galactomannan from locust bean and a glucomannan from konjac tuber

The synergistic interaction between three red algae extracts and the galactomannan from locust bean (Ceratonia siliqua L.) and the glucomannan from the konjac tuber (Amorphophallus konjac C. Koch (syn.A. rivieri Durien var. konjac (C. Kock) Engler)) has been characterized in terms of gel properties. The extract obtained fromEucheuma alvarezii Doty (E. cottonii of commerce) was highly synergistic with bothkonjac flour and locust bean gum.Furcellaria fastigiata (Huds.) Lamour andEucheuma gelatinae (Esper) extracts were only slightly synergistic with locust bean gum, but were found to be highly synergistic with konjac flour.     

Branch,micropropagule and tissue culture of the red algaeEucheuma denticulatum andKappaphycus alvarezii farmed in the Philippines

Three forms of the iota-producing carrageenophyte,Eucheuma denticulatum, and four forms of the kappa-producing carrageenophyte,Kappaphycus alvarezii, obtained from seaweed farms in the Philippines have been grown in the laboratory under unialgal and axenic conditions. Comparison of media indicates that seed stocks of both species can be cultured using enriched seawater media ranging from ESS and SWMD-1 to inexpensive soil extract (Erdshreiber's) or holding in sterile seawater for up to three weeks. Micropropagation has been successful with at least two forms of each species resulting in clonal propagation from axenic explants within 4 to 8 weeks. Callus development and branch regeneration has also been induced in two forms of each species. The results indicate that culture facilities in the farming areas of the Philippines could maintain high-yielding and rapidly growing seed stock for the seaweed farmers.     

Systematics and genetic variation in commercial shape Kappaphycus and shape Eucheuma (Solieriaceae, Rhodophyta)

The systematics and taxonomy of Kappaphycus and Eucheuma (Solieriaceae) is confused and difficult due to morphological plasticity, lack of adequate characters to identify species and commercial names of convenience. These taxa are geographically widely dispersed through cultivation. Commercial, wild and herbarium sources were analysed; molecular markers provided insights into taxonomy and genetic variation, and where sources of genetic variation may be located. The mitochondrial cox2-3 and plastidal RuBisCo spacers were sequenced. There is a clear genetic distinction between K. alvarezii (“cottonii”) and K. striatum (“sacol”) samples. Kappaphycus alvarezii from Hawaii and some samples from Africa are also genetically distinct. Our data also show that all currently cultivated K. alvarezii from all over the world have a similar mitochondrial haplotype. Within Eucheuma denticulatum (“spinosum”) most African samples are again genetically distinct. Our data also suggest that currently cultivated E. denticulatum may have been “domesticated” several times, whereas this is not evident for the cultivated K. alvarezii. The present markers used do not distinguish all the morpho-types known in cultivation (e.g. var. tambalang, “giant” type) but do suggest that these markers may be useful to assess introductions and species identification in samples.     

Factors Influencing the Growth Rates of Three Commercial Eucheumoids at Coastal Sites in Southern Kenya

As a possible means of improving the livelihoods of local villagers, off-bottom rope cultivation of commercial eucheumoids was investigated on the southern Kenyan coast at three sites, representative of the variety of environments. The morphotypes used were brown Eucheuma denticulatum and green and brown Kappaphycus alvarezii. The study was carried out over a 15 month period from August 2001 until October 2002. Relative growth rates were highest at a sandy flat in a mangrove system (Gazi; 5.6% d⁻¹), and lowest in an intertidal reef flat (Kibuyuni; 3.2% d⁻¹) with a lagoon being intermediate (Mkwiro; 4.8% d⁻¹). The brown E. denticulatum had the highest growth rate of 4.7% d⁻¹ compared to the green and brown K. alvarezii which were 4.3% d⁻¹ and 4.2% d⁻¹, respectively. Growth was more variable at Kibuyuni and Mkwiro. The growth was higher during the southeast monsoon (4.7% d⁻¹) than during the northeast monsoon (4.0% d⁻¹). This is part of a larger study and the effects of water motion, salinity, temperature, thallus nitrogen, and ‘ice-ice’ syndrome on growth of morphotypes is discussed. The water motion was observed to increase thallus nitrogen and hence the growth of eucheumoids. The ‘ice-ice’ condition affected both brown E. denticulatum and brown K. alvarezii but not green K. alvarezii. The results suggest that commercial cultivation of eucheumoids in Kenya will be feasible.     

Evaluation of ethanol production and bioadsorption of heavy metals by various red seaweeds

The objective of this study was to evaluate ethanol production and bioadsorption with four red seaweeds, Gelidium amansii, Gracilaria verrucosa, Kappaphycus alvarezii and Eucheuma denticulatum. To produce ethanol, thermal acid hydrolysis, enzymatic saccharification and fermentation was carried out. After pretreatment, 38.5, 39.9, 31.0 and 27.5 g/L of monosaccharides were obtained from G. amansii, G. verrucosa, K. alvarezii and E. denticulatum, respectively. Ethanol fermentation was performed with Saccharomyces cerevisiae KCCM 1129 adapted to 80 g/L galactose. The ethanol productions by G. amansii, G. verrucosa, K. alvarezii and E. denticulatum were 18.8 g/L with Y _EtOH = 0.49, 19.1 g/L with Y _EtOH = 0.48, 14.5 g/L with Y _EtOH = 0.47 and 13.0 g/L with Y _EtOH = 0.47, respectively. The waste seaweed slurries after the ethanol fermentation were reused to adsorb Cd(II), Pb(II) and Cu(II). Using langmuir isotherm model, Cu(II) had the highest affinity for waste seaweeds with the highest q _max and electronegativity values among three heavy metals.     

Differences in biomass production and carrageenan yields among four strains of farmed carrageenophytes in Northern Bohol,Philippines

Comparative studies on the biomass and carrageenan production of two strains ofEucheuma denticulatum and two strains ofKappaphycus alvarezii were made to assess the seasonality in their production capacities.The high and similar refined carrageenan (RC) yields (43–53% of dry wt.) of the four strains in the first cropping season (June–October) coincided with their high biomass production with plants averaging from 1.1 to 1.8 kg each at harvest. The poor RC yields (21–33%) recorded in the second cropping (October–February) coincided with their season of low biomass (av. wt: 0.34 to 1.0 kg). The four strains, however, recorded contrasting performance in the third cropping season (February–July) with the twoE. denticulatum strains recording high RC yields (43 and 42.5%) together with high biomass (av. wt: 1.5 and 1.6 kg) in contrast to the low RC yields (30 and 39%) and low biomass (av. wt. 0.21 and 0.28 kg) of the twoK. alvarezii strains. Records for semi-refined carrageenan (SRC) yields in the second and third cropping seasons were quite consistent and similar for the four strains (42–55%), except in the second cropping where the twoK. alvarezii strains recorded low SRC. These differences in production potentials highlight the need for cropping management of the four strains to improve their cropping performance.     

Phylogeny of Betaphycus (Gigartinales,Rhodophyta) as inferred from COI sequences and morphological observations on B. philippinensis

The phylogeny of the three species that comprise the genus Betaphycus Doty in Silva, Basson and Moe and their phylogenetic relationships with other eucheumatoids are still unresolved. In this study, the utility of the mitochondrial cytochrome oxidase I (COI) marker in resolving their relationships was evaluated. Analyses of the COI sequences from Betaphycus philippinensis Doty and Betaphycus speciosus (Sonder) Doty ex Silva specimens collected from their type localities (Sorsogon, Philippines and Western Australia, respectively) revealed that the two species formed a well-supported clade distinct from Eucheuma J. Agardh and Kappaphycus Doty ex Silva. The genotyped specimens of B. philippinensis were observed to exhibit dorsal protuberances, a characteristic which has been regarded as a key diagnostic feature of Betaphycus gelatinus (Esper) Doty ex Silva. This observation raised the possibility that these two taxa are conspecific. In addition, B. philippinensis specimens were also observed to exhibit morphological features that could be used to distinguish the species from other eucheumatoids, such as the location tetrasporangial nemathecium in the thallus and the presence of apical or lateral pit connections in the tetraspores. The species referred to in the literature as “B. gelatinus” (as Eucheuma gelatinae) collected from northwestern Philippines was identified as a species of Eucheuma based on molecular and morphological evidence. The phylogenetic relationships of this species with other related eucheumatoid taxa were also determined.     

Laboratory and field growth studies of commercial strains ofEucheuma denticulatum andKappaphycus alvarezii in the Philippines

Daily growth rates of 0.1 to 8.4% d^-1 for the brown form and 0.2 to 6.3% d^-1 of the green form were measured for 3 to 5-cm long branches of the tropical red seaweedKappaphycus alvarezii cultured in the laboratory. Highest growth rates were found using inexpensive enrichments such as soil water and coconut water supplemented with 0.7 mM N and 13 µM P and with a liquid fertilizer, Algafer, produced from seaweeds in the Philippines. Laboratory grown branches of bothK. alvarezii andEucheuma denticulatum transplanted to rafts in the field showed daily growth rates of 4.4 to 8.9% d^-1, as high or higher than other reported growth rates. The studies, carried out in the Philippines, demonstrate the viability and high yield of laboratory cultivars and methods to keep laboratory culture costs low.     

Introduction of non-native species of Kappaphycus (Rhodophyta, Gigartinales) in subtropical waters: Comparative analysis of growth rates of Kappaphycus alvarezii and Kappaphycus striatum in vitro and in the sea in south-eastern Brazil

We compared the growth rates of Kappaphycus alvarezii (Doty) Doty ex P. Silva and Kappaphycus striatum (Schmitz) Doty, both, in vitro under different conditions of light and temperature, and in the sea. Temperature was the most important factor controlling the growth of both species, in vitro and in the field. In the sea there was a clear seasonal pattern in growth rate, attributed to temperature and salinity variation. The lower growth rates were registered in winter and spring, and the highest in summer and autumn months for both species. Based on growth rate in the field, and the production of viable tetraspores during the summer in Kappaphycus striatum, we conclude that is more profitable, and ecologically safer, to only continue with the introduction program of Kappaphycus alvarezii.     

Phylogenetic relationship of Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae, Rhodophyta) in Malaysia

The genera Kappaphycus Doty and Eucheuma J. Agardh are important sources of carrageenan in Malaysia, offering lucrative revenues to the carrageenan industry, economy, and the local community. The extensive range of morphotypes and the lack of distinct morphological characteristics led to the application of molecular systematics in elucidating this taxonomic confusion. Local varieties of Kappaphycus and Eucheuma, identified using putative external morphology, were analyzed using the mitochondrial cox2–3 spacer and plastid RuBisCO spacer molecular markers. Phylogenetic analysis of these and non-local specimens indicate that Kappaphycus and Eucheuma are genetically distinct. Three main genotypes of Kappaphycus alvarezii were identified, of which two are extant in Hawaii. Morphological and color variations are not supported by molecular data, indicating that most of the local names are not genetically based. Both the cox2–3 spacer and RuBisCO spacer generated phylogenetic trees with similar topology except in variation of nodal supports. The two markers showed clear separation between Kappaphycus and Eucheuma and the existence of three Malaysian Kappaphycus cultivars. Cox2–3 spacer data is more variable and provides better resolution than the RuBisCO spacer, showing that Kappaphycus is more diversified with a larger number of genotypes, strains, and species which are unique to Southeast Asia. Kappaphycus sp. “Aring-aring” appeared to be phenotypically and genotypically different from other Kappaphycus congeners, whereas Kappaphycus striatum exhibited two different genotypes. Our data indicate that Eucheuma denticulatum is the dominant species in Malaysian waters and also suggested paraphyly in Eucheuma which will require further studies. The application of molecular taxonomy on Malaysian Kappaphycus and Eucheuma proves useful, offering valuable insights into the taxonomy and distribution of these commercially important Rhodophytes.     

Occasional pathogenic bacteria promoting ice-ice disease in the carrageenan-producing red algae Kappaphycus alvarezii and Eucheuma denticulatum (Solieriaceae,Gigartinales, Rhodophyta)

The bacterial isolates from normal and diseased branches of Kappaphycus alvarezii and Eucheuma denticulatum in the Philippines were examined for possible role in the development of the ice-ice disease. The numbers of bacteria on and in ice-iced branches were 10–100 times greater than those from normal, healthy ones. Gram-positive bacteria predominated in almost all branch sources, but with an increasing proportion of agar-lysing bacteria in branches suffering from the ice-ice disease. These agar-lysing bacteria were composed of yellow and non-pigmented, spreading colonies identified to the Cytophaga-Flavobacterium complex and the Vibrio group. Among isolates which mainly appeared on ice-iced branches, two strains, designated as P11 (Vibrio sp.) and P25 (Cytophage sp.), which showed pathogenic activity, were obtained. These strains caused early ice-ice whitening of K. alvarezii especially when subjecting branches to environmental stress, such as reduced salinity and light intensity, suggesting that these bacteria were occasionally pathogenic. This paper offers new evidence of bacterial role in the development of so-called ice-ice disease among farmed species of Kappaphycus.     

Production of clonal planting materials from Gracilaria changii and Kappaphycus alvarezii through tissue culture and culture of G. changii explants in airlift photobioreactors

Global demand for seaweed resources has increased due to their emergent use as sources of biopharmaceuticals, nutraceuticals and biofuels. These high-valued products make possible the use of micropropagation techniques that may be more costly than conventional mariculture. This study reports the successful tissue culture of Kappaphycus alvarezii (Doty) Doty ex P. C. Silva and Gracilaria changii (B. Xia & Abbott) Abbott, Zhang and Xia. Callus induction of K. alvarezii was successfully developed following an explant sterilisation protocol. Callus formation and regeneration of K. alvarezii was observed in solidified Provasoli’s enriched seawater medium. Different culture conditions such as agar concentration, growth hormones, nutrients, irradiance and enrichment media were investigated to determine the suitable conditions for explant culture of G. changii. Proliferations of adventitious shoots were induced under the most suitable culture conditions. G. changii explants were successfully cultured in airlift photo-bioreactors, with no decrease in the carbohydrate content in the G. changii explants. This micropropagation technique can provide a useful alternative system for seedling production of economically important seaweeds.     

Investigation of the application of Acadian Marine Plant Extract Powder (AMPEP) to enhance the growth, phenolic content, free radical scavenging, and iron chelating activities of Kappaphycus Doty (Solieriaceae, Gigartinales, Rhodophyta)

An optimization study on concentration (viz. 0.01, 0.1, and 1.0 g L^?1) and dipping time (i.e., 30 and 60 min) was conducted on three different color morphotypes (i.e., reddish brown, yellowish brown and purple) of the commercial carrageenophyte Kappaphycus alvarezii (Doty) Doty. The study tested the efficacy of Acadian Marine Plant Extract Powder (AMPEP) on the growth rate and occurrence of macro-epiphytes from August to November, representing the wet season of the Philippines. The optimum concentration and dipping time were obtained at 0.1 g L^?1 and 30 min, respectively. These optimum parameters were then further verified in a commercial nursery using the yellowish brown morphotype. In another experiment, K. alvarezii (tambalang purple morphotype) and Kappaphycus striatum (Schmitz) Doty (sacol green morphotype) with, and without, AMPEP dippings were tested for their total phenolic content, free radical scavenging and iron chelating activities. Seaweed dipped in AMPEP demonstrated higher growth rates than the control. Lower concentrations (i.e., 0.01-0.1 g L^?1) and shorter dipping time (e.g., 30 min) produced higher growth rates than the highest concentration (1.0 g L^?1) and longer (60 min) dipping time. The presence of macro-epiphytes such as filamentous Ulva did not adversely affect the robust growth of the three color morphotypes of K. alvarezii. The lowest and highest growth rates obtained in a commercial seaweed nursery using the optimum concentration and dipping time of AMPEP were observed in July and January with 0.8% and 6.7% day^?1, respectively. The antioxidant content of K. alvarezii (tambalang purple) and K. striatum (sacol green) responded differently to AMPEP dipping. The changes in total antioxidant activity followed almost the same trend as in phenolic content, in both K. alvarezii (tambalang purple) and K. striatum (sacol green), whereas, the iron chelating ability of both seaweeds with and without AMPEP dipping varied monthly. The results obtained for the use of AMPEP dips for commercial Kappaphycus cultivation demonstrated an effective management tool for improved farming protocols.     

The farmed Eucheuma species (Gigartinales,Rhodophyta) in Danajon Reef,Philippines: carrageenan properties

Six cultured strains of Eucheuma denticulatum and E. alvarezii, from which stocks can be selected for the development of a Eucheuma seedling bank, were tested for their carrageenan quality from June to November 1988. Percent yield of all the varieties taken together was apparently higher in June, becoming lower in November (regression, r –0.785, probability, p 0.001). Stepwise regression analysis was done to determine the existence of any relationship between any of the following parameters: gel strength, viscosity, sulfate content, month of sampling, and yield, whether taken individually or in combination. Results show variations of the yield with the month of sampling. ANOVA was performed to test whether there are differences in sulfate levels, gel strength, and viscosity between the Eucheuma alvarezii morphotypes. There was no significant difference between the green and the brown types.