Carrageenophytes of occidental Portuguese coast: 1-spectroscopic analysis in eight carrageenophytes from Buarcos bay

Infrared and Raman spectroscopic analysis of the carrageenan (alkaline extraction) in eight species (representing seven genera and four families) of Gigartinales, in different reproductive phases from Buarcos bay (Figueira da Foz, Portugal), were studied. Female gametophytes and non-fertile thalli samples of Chondrus crispus, Mastocarpus stellatus, Chondracanthus teedei var. lusitanicus, Gigartina pistillata and Chondracanthus acicularis present a kappa-carrageenan profile or varying degrees of a kappa-iota hybrid. The presence of kappa-iota hybrid carrageenan in C. teedei var. lusitanicus was confirmed by 13C NMR. The carrageenans extracted from Gymnogongrus crenulatus and Ahnfeltiopsis devoniensis are constituted mainly by iota-carrageenan but seasonal variations in the nature of carrageenans are present. lambda-Family carrageenans were found in tetrasporophytes of C. crispus, M. stellatus, C. teedei var. lusitanicus, C. acicularis and G. pistillata. Calliblepharis jubata presents carrageenans of iota-type in all reproductive stages.     

Immunochemistry of kappa-type carrageenans from certain red algae

Carrageenans from female and male gametophytic plants of the alga Rhodo-glossum californicum, female plants of Chondrus crispus and Gigartina pistillata, and male plants of Iridaea cordata and a Gigartina species from San Francisco Bay were fractionated into potassium chloride-soluble and -insoluble components and were analysed chemically. An anti-K-carrageenan, the reactivity of which is directed to K-type structures (i.e., 3-linked d-galactose 4-sulphate and 4-linked 3,6-anhydro-D-galactose residues) was used to analyse these carrageenans immunochemically. The potassium chloride-insoluble carrageenans from these species were found to be highly reactive K-type carrageenans. The potassium chloride-soluble carrageenans were less reactive to anti-K-carrageenan and, in addition, showed reactivity to an anti-λ-carrageenan preparation. The chemical and immunochemical data suggest that the potassium chloride-soluble carrageenans contain either λ- or μ-carrageenan, as a high proportion of the precursors to the 3,6-anhydro-D-galactose are 4-linked D-galactose 2,6-disulphate residues, and no increase in immunological reactivity to anti K-carrageenan was observed upon alkali treatment.     

LIFE HISTORY PHASES AND THE BIOMECHANICAL PROPERTIES OF THE RED ALGA CHONDRUS CRISPUS (RHODOPHYTA)

Chondrus crispus Stackhouse alternates between two isomorphic life history phases that differ in cell-wall phycocolloid composition. It has been long hypothesized that the gametophyte, with strong-gelling kappa-type carrageenans, is mechanically superior to the tetrasporophyte, with nongelling lambda-type carrageenans, which could contribute to the observed gametophytic dominance in many wave-swept environments. Standard mechanical tests were performed on distal tissues of C. crispus sampled from a range of environments in Narragansett Bay, Rhode Island, using a tensometer equipped with a video extensometer. Life history phase was by far the most important determinant of mechanical properties, whereas environmental factors had only modest influence (vertical distribution) or no effect (exposure); gametophytic distal tissues were 43% stronger, 21% more extensible, and 21% stiffer than tetrasporophytic distal tissues. However, the superior strength of gametophytic tissues was not evident at the stipe/holdfast junction (where breakage typically occurs), and the two phases were equally susceptible to dislodgment by a given force. The primary ecophysiological role of carrageenans in C. crispus may not be the provision of a structure to resist wave action.     

Heterogeneity of carrageenans from Chondrus crispus.

Carrageenans extracted from gametophytic and sporophytic Chondrus crispus were analysed by hydrolysis, KCl fractionation and 1H NMR spectroscopy. The carrageenan from gametophytic plants is composed predominantly of two KCl insoluble fractions which contain kappa-carrageenan as the major component with 1-carrageenan and sulphated galactans as minor components. The precursor mu- and v-carrageenans were not found in the soluble fraction. The extract from sporophytic plants is composed mainly of a KCl soluble fraction which could be separated into 10 fractions by ion-exchange chromatography. The major component did not show a lambda-type structure but one of a xi-carrageenan.     

The chemistry and immunochemistry of carrageenans from Eucheuma and related algal species

Carrageenans from several species of Eucheuma have been fractionated into KCI-soluble and KCI-insoluble fractions and analyzed by the usual chemical procedures. An anti-K-carrageenan, the reactivity of which is directed to K-structures (i.e., 3-linked galactose 4-sulphate, and 4-linked 3,6-anhydrogalactose) was used to analyze these carrageenans immunochemically. The antibody preparation shows only a small amount of cross-reactivity with i-type carrageenans and thus could be used to distinguish K- and i-type carrageenans, the latter having an index of homology of less than 0.2. A comparison of chemical and immunochemical data yielded further information as to the nature of the carrageenan-anti-carrageenan interaction, as well as elucidating the finer structure of carrageenans.     

On the structure of kappa/iota-hybrid carrageenans

The coil-to-helix transition and temperature dependence of the viscosity of commercial kappa/iota-hybrid carrageenans produced by the red algae Sarcothalia crispata, Mazaella laminarioides, and Chondrus crispus were studied using rheometry and optical rotation. The structure of these kappa/iota-hybrid carrageenans was determined by 1H and 13C NMR spectroscopy combined with monosaccharide composition analysis. The coil-to-helix transitions, measured by polarimetry and rheometry, of the kappa/iota-hybrid carrageenans are significantly different from those of pure kappa- and iota-carrageenan, and from hand-made mixtures thereof. This provides evidence that the kappa/iota-hybrid carrageenans are mixed chains, containing both kappa- and iota-repeating units.     

Apoplastic oxidation of L-asparagine is involved in the control of the green algal endophyte Acrochaete operculata Correa & Nielsen by the red seaweed Chondrus crispus Stackhouse

Gametophytes of the marine alga Chondrus crispus are more resistant than tetrasporophytes to infection by the filamentous endophytic alga Acrochaete operculata. It has been shown recently that carrageenan oligosaccharides from the resistant gametophytic generation of C. crispus stimulate the secretion of L-asparagine (L-Asn) by the endophyte and that the host generates hydrogen peroxide and 2-oxo-succinamic acid after contact with this amino acid. Here the response of C. crispus to L-Asn and its effect on the pathogen is investigated. Chondrus crispus released hydrogen peroxide, ammonium ions, and a carbonyl compound into the medium when exposed to L-Asn. This response was correlated with an increase in oxygen consumption. Inhibitor studies indicated the involvement of a flavoenzyme in the reaction, which was sensitive to high concentrations of the reaction product, ammonium, and to chlorpromazine, quinacrine, and cyanide, inhibitors of L-amino acid oxidase. Cell wall macerate of C. crispus also responded to L-Asn, while protoplasts were inactive. Uptake of L-Asn into the cell was not necessary for the response, suggesting that the involved L-amino acid oxidase is apoplastic. Acrochaete operculata was more sensitive to hydrogen peroxide than C. crispus and settlement of A. operculata zoospores on C. crispus was reduced by 86% in the presence of L-Asn. This reduced settlement could be prevented with catalase. Chondrus crispus thus features an apoplastic amino acid oxidase, which is involved in the control of its endophytic pathogen. The modulation of the amino acid secretion in A. operculata by carrageenan oligosaccharides is therefore a key issue in the etiology of the association.     

Stereoselective synthesis of [13C]methyl 2-[15N]amino-2-deoxy-beta-D-glucopyranoside derivatives

The chemical structure of carrageenans produced by the gametophytic and tetrasporophytic life cycle phases of Gigartina pistillata has been determined by permethylation analysis, IR and 13C NMR spectroscopies. The chemistry of the galactans varies according to the biological phases of the plant, the gametophytic alga produces heterogeneous kappa-iota type carrageenan containing minor amounts of nu-carrabiose. The tetrasporophytic alga synthesizes a complex sulfated galactan composed of lambda-, xi-, pi-carrabioses and sulfated carrabioses containing 3-linked galactopyranose 2,6-disulfate.     

DIFFERENTIATION OF LAMBDA CARRAGEENANS FROM RHODOPHYTA WITH IMMUNOLOGICAL AND SPECTROSCOPIC TECHNIQUES1

KCl soluble carrageenans from sporophytes of two Gigartina species were differentiated from those of Chondrus crispus Stackhouse, Iridaea cordata (Turner) Bory, Rhodoglossum californicum (J. Ag.) Abbott, and two Petrocelis species by their failure to precipitate on immunodiffusion against either of two absorbed anti-λ–carrageenan antisera. The carrageenans have been analyzed by infrared spectroscopy, and on this basis it is postulated that the antibody preparations recognize sulphate on C-6 of the carrageenans. The Gigartina carrageenans appear to lack sulfate at this position.     

Influence of tissue source and growth rates on dry weight and carrageenan composition of Chondrus crispus (Gigartinales,Rhodophyta)

Carrageenan analyses were conducted on vegetative female clones of Chondrus crispus that were cultured to provide tissues with differing growth rates. Tissue dry weights increased from apex to base of fronds. Total carrageenan contents were lower in apical 1 to 2 cm segments than elsewhere in the frond, except when the alga was grown at high photon irradiances. Clone 373A contained more carrageenan than clone G8. The proportion of 0.3 M KCl-soluble polymers in the total native carrageenans varied from 44 to 92%, being highest in older tissues of fronds cultured at high photon irradiances. The apical 1 cm segments contained less KCl-soluble carrageenans than other tissues from the corresponding fronds. The KCl-soluble carrageenans, when alkali-modified and refractionated, afforded the expected kappa-iota carrageenan in > 79% yields. The remainder consisted of a polymer containing 23.1% SO₃Na and 8.4% 3,6-anhydrogalactose. Lambda carrageenan was not detected. Variations in carrageenan distribution between the apical region and other parts of the frond may reflect the increasing influence of medullary tissue developed as the immature cells differentiate.     

Low molecular weight derivatives of different carrageenan types and their antiviral activity

A number of low molecular weight (LMW) fractions of carrageenans with different structural types were obtained by free radical depolymerization (H₂O₂), mild acid hydrolysis (HCl), and a specific enzyme. Three samples of carrageenans were depolymerized: kappa-carrageenan from Chondrus armatus, kappa-carrageenan from Kappaphycus alvarezii, and kappa/beta-carrageenan from Tichocarpus crinitus with initial molecular weights of 250, 390, and 400 kDa, respectively. The chemical depolymerization by two methods resulted to LMW derivatives of carrageenans with molecular weight from 1.2 to 3.5 kDa. Oligosaccharides of kappa- and kappa/beta-carrageenans with molecular weight of 2.2 and 4.3 kDa, respectively, were obtained after enzymatic depolymerization by recombinant kappa-carrageenase from Pseudoalteromonas carrageenovora. It was shown that the antiviral activity of high molecular weight carrageenans against tobacco mosaic virus was higher than that of their LWM derivatives independently on the depolymerization method. The method of depolymerization had some influence on the antiviral activity of carrageenan. LMW derivatives of kappa- and kappa/beta-carrageenans obtained by mild acid hydrolysis showed higher antiviral activity than the products of free radical depolymerization. The oligosaccharides prepared by enzymatic degradation possessed the lowest activity.     

Seasonal variations in thalli and carrageenan composition of Gigartina pistillata (Gmelin) Stackhouse (Rhodophyta, Gigartinales) harvested along the Atlantic coast of Morocco

The biology and biochemistry of Gigartina pistillata (Gmelin) Stackhouse collected monthly at Nation Beach (Morocco), was studied during one year. The biological study showed one period of active growth from April to July. The thallus composition was quite stable during the major part of the year. The dry matter was maximum in May and August and minimum in January. The maximum carrageenan content occurred in June and September (about 37%) and the minimum carrageenan content occurred in February (19.0%). The total nitrogen content varied significantly, with a maximum in January (1.98%) and a minimum in August (0.7%). The ash content was significant (23–32%) with a maximum in August and a minimum in May. The carrageenan extracted from natural populations of Gigartina pistillata was a mixture of lambda‐type and kappa‐type carrageenans. The 3,6‐anhydrogalactose varied between 4.5 mol% in June to 25 mol% in February. For industrial applications the extract could be considered as a lambda‐type. The best period for harvest of G. pistillata in Morocco is between July and August when biomass and viscosity are at their maximum. A relationship between the physical characteristics of G. pistillata carrageenans and its seasonal cycle was deduced.     

A comparative analysis of phycocolloids produced by underutilized versus industrially utilized carrageenophytes (Gigartinales, Rhodophyta)

Carrageenan (E-407) and semi-refined carrageenan (E-407a) are some of the main additives used by the food industry for their gelling, emulsifying, thickening, and stabilizing properties. These are natural ingredients, which have been used for decades in food applications and are generally regarded as safe. Internationally, sub-tropical carrageenophytes (e.g., Kappaphycus alvarezii) are cultivated extensively as a source of raw materials for industrial extraction, and their use as potential candidates in integrated multitrophic aquaculture is tentative. We analyzed carrageenan yield (as a percentage of dry weight) and chemical composition (by Fourier transform infrared attenuated total reflection and Fourier transform-Raman) of extracts produced by several carrageenophytes (Gigartinales, Rhodophyta), from different origins, e.g., K. alvarezii (Tanzania, Indonesia, the Philippines, Panama, and Mexico), Kappaphycus striatum (Madagascar), Eucheuma denticulatum (Tanzania, the Philippines, and Madagascar), Betaphycus gelatinum (the Philippines), Chondracanthus chamissoi, and Sarcothalia crispata (Chile). For comparison, some underutilized carrageenophytes were also analyzed, e.g., Chondrus crispus, Mastocarpus stellatus, Gigartina pistillata, Chondracanthus teedei var. lusitanicus, Chondracanthus acicularis, Calliblepharis jubata, Gymnogongrus crenulatus, and Ahnfeltiopsis devoniensis (Portuguese carrageenophytes). The main findings were that the highest carrageenan yield was obtained from K. striatum (Madagascar) with 75.6 (percent dry weight (% DW)); B. gelatinum and K. alvarezii (both from the Philippines) had yields of 71.0% and 68.0% (% DW), respectively; and G. pistillata (Portugal) 65.4% (% DW). Spectroscopic analysis of the phycocolloids allowed determination of a wide range of carrageenan types, e.g., pure iota carrageenan, several kappa–iota hybrid carrageenans with different iota/kappa ratios, and kappa–beta, xi–theta, and xi–lambda hybrid carrageenans.     

CO-OCCURRENCE OF CARRAGEENANS AND AGAROIDS IN GYMNOGONGRUS TORULOSUS (GIGARTINALES, PHYLLOPHORACEAE) FROM ARGENTINA

The red seaweeds, Gigartina skottbetgii and Sarcothalia crispata , have commercial value as raw material for the industrial production of phycocolloids (carrageenans) in Argentina. The third alga with potencial possibilities for the carrageenan production is Gymnogongrus torulosus. Herein, we report the study of the polysaccharide system present in Gymnogongrus torulosus , which contribute to the estimation of the importance of this algae in the seaweed industry. Analysis of the hot water-soluble extract (C1), by FT-IR, methylation and ¹³C NMR, showed mainly the presence of iota/kappa carrageenan hybrid (molar ratio ∼ 2:1). The soluble fractions obtained after KCl fractionation (F3, 16.1 % of C1) and soluble after alkaline treatment and KCl fractionation (F3T3, 34.6% of F3T) gave negative optical rotations (−15.5 and −55.4, respectively), considerably lower than those reported for kappa/iota carrageenans (from 56.1 to 66.5). These fractions (F3 and F3T3) have significant quantities of L-galactose (11.1% and 29.8%) and L-3,6-anhydrogalactose (19.2% and 4.3%). The direct relationship between the optical rotation and the percentage of L-galactose indicated that its structural influence is similar in all the fractions. The results suggest that Gymnogongrus torulosus biosynthesizes a polysaccharide system with co-occurrence of carrageenans and agaroids in the same thallus.     

Sulfated Oligosaccharides Mediate the Interaction between a Marine Red Alga and Its Green Algal Pathogenic Endophyte.

The endophytic green alga Acrochaete operculata completely colonizes the sporophytes of the red alga Chondrus crispus; however, it does not penetrate beyond the outer cell layers of the gametophytes. Given that the life cycle phases of C. crispus differ in the sulfation pattern of their extracellular matrix carrageenans, we investigated whether carra-geenan fragments could modulate parasite virulence. lambda-Carrageenan oligosaccharides induced release of H(2)O(2), stimulated protein synthesis, increased carrageenolytic activity, and induced specific polypeptides in the pathogen, resulting in a marked increase in pathogenicity. In contrast, kappa-carrageenan oligosaccharides did not induce a marked release of H(2)O(2) from A. operculata but hindered amino acid uptake and enhanced their recognition by the host, resulting in a reduced virulence. Moreover, C. crispus life cycle phases were shown to behave differently in their response to challenge with cell-free extracts of A. operculata. Gametophytes exhibited a large burst of H(2)O(2), whereas only low levels were released from the sporophytes.     

Composition and distribution of carrabiose moieties in hybrid kappa-/iota-carrageenans using carrageenases

Hybrid kappa/iota-carrageeans extracted from Gigartina skottsbergii, Chondracanthus chamisso?, and Chondrus crispus were incubated with Pseudoalteromonas carrageenovora kappa-carrageenase and Alteromonas fortis iota-carrageenase. The degradation products as well as the resistant fraction were fully characterized by chromatography, NMR, and mass spectrometry. The low percentage of degradation observed after treatment by the iota-carrageenase suggests that long segments of iota-carrabiose or block of iota-carrageenan are low in abundance. The degradation products of the kappa-carrageenase allow discriminating three modes of kappa-carrabiose distribution: blocks of kappa-carrabiose, kappa-carrabiose rich fraction containing iota-carrabiose units probably randomly distributed, and iota-carrabiose rich fraction which corresponds to the resistant fraction. The proportions of each fraction were related to the botanical origin as well as the place of growth of the seaweed.     

FOURIER TRANSFORM INFRARED MICROSPECTROSCOPY AS A TOOL TO IDENTIFY MACROALGAL PROPAGULES1

Understanding of macroalgal dispersal has been hindered by the difficulty in identifying propagules. Different carrageenans typically occur in gametophytes and tetrasporophytes of the red algal family Gigartinaceae, and we may expect that carpospores and tetraspores also differ in composition of carrageenans. Using Fourier transform infrared (FT‐IR) microspectroscopy, we tested the model that differences in carrageenans and other cellular constituents between nuclear phases should allow us to discriminate carpospores and tetraspores of Chondrus verrucosus Mikami. Spectral data suggest that carposporophytes isolated from the pericarp and female gametophytes contained κ‐carrageenan, whereas tetrasporophytes contained λ‐carrageenan. However, both carpospores and tetraspores exhibited absorbances in wave bands characteristic of κ‐, ι‐, and λ‐carrageenans. Carpospores contained more proteins and may be more photosynthetically active than tetraspores, which contained more lipid reserves. We draw analogies to planktotrophic and lecithotrophic larvae. These differences in cellular chemistry allowed reliable discrimination of spores, but pretreatment of spectral data affected the accuracy of classification. The best classification of spores was achieved with extended multiplicative signal correction (EMSC) pretreatment using partial least squares discrimination analysis, with correct classification of 86% of carpospores and 83% of tetraspores. Classification may be further improved by using synchrotron FT‐IR microspectroscopy because of its inherently higher signal‐to‐noise ratio compared with microspectroscopy using conventional sources of IR. This study demonstrates that FT‐IR microspectroscopy and bioinformatics are useful tools to advance our understanding of algal dispersal ecology through discrimination of morphologically similar propagules both within and potentially between species.     

CARRAGEENANS BIOSYNTHESIZED BY CARPOSPOROPHYTES OF RED SEAWEEDS GIGARTINA SKOTTSBERGII (GIGARTINACEAE) AND GYMNOGONGRUS TORULOSUS (PHYLLOPHORACEAE)1

Carrageenans biosynthesized by gametophytic and tetrasporic plants of seaweeds belonging to the Gigartinaceae and Phyllophoraceae are different: gametophytes produce carrageenans of the kappa family, whereas lambda‐carrageenans are extracted from tetrasporophytes. For Gigartina skottsbergii Setchell and Gardner and Gymnogongrus torulosus Hooker et Harvey, mature cystocarps were isolated and carrageenans were extracted. Structural determination by methylation analysis, Fourier transform infrared spectroscopy, and ¹³C‐NMR spectroscopy showed that they were kappa/iota‐carrageenans. For the extract obtained from cystocarps of Gigartina skottsbergii with water at room temperature, the ratio kappa:iota was 1:0.30 and at 90° C was 1:0.43; significant amounts of precursors were also present. The extract obtained from cystocarps of Gymnogongrus torulosus at 90° C showed prevalence of iota‐carrageenans (ratio kappa:iota 1:1.21). These extracts are similar to the polysaccharides produced by gametophytes of these seaweeds. For Gigartina skottsbergii, it was possible to separate the pericarpic tissue from the carposporophyte. Thus, they were extracted separately, and the carrageenans isolated were studied as described before, obtaining similar conclusions. These results clearly show that whereas the carposporophytes are located inside the cystocarp, they produce carrageenans of the kappa family despite of being diploid cells.     

Carrageenans from Cystocarpic and Sterile Plants of Chondrus Pinnulatus (Gigartinaceae, Rhodophyta) Collected from the Russian Pacific Coast

The chemical structure, gel properties and biological activity of the carrageenans isolated from cystocarpic and sterile plants of Chondrus pinnulatus were investigated. The total carrageenan content of the sterile plant was observed to be twice that of the cystocarpic plants. According to data obtained by ¹³C-NMR and FT IR, the gelling polysaccharides from cystocarpic and sterile plants of C. pinulatus have similar structures and were identified as κ/ι-carrageenans. The difference between these polysaccharides was in the ratio of the κ- and ι-segments, with a predominant content of κ-segments in cystocarpic plants (80%). Moreover, KCl-insoluble fractions possibly contain hetero-disperse μ/ν precursor: amounts of this in the polysaccharide from sterile plants were more than that extracted from the cystocarpic plants. The KCl-soluble fractions (non gelling) were λ-carrageenans with another carrageenan type that had a low amount of 3,6-anhydrogalactose. Carrageenans from cystocarpic stages showed good gelling properties, whereas those from sterile plants formed a very weak gel. Structural differences and molecular weight of carrageenans obviously determine the biological activity of the polysaccharides. Non gelling-carrageenans from both types of ιt C. pinnulatus plants showed high macrophage-phosphatase activity and κ/ι-carrageenan from cystocarpic plant possessed a potent anti-coagulant activity, which was extremely strong in a low concentration of 100 μg ml⁻¹.     

The production of antimicrobial compounds by British marine algae I. Antibiotic-producing marine algae

Using five species of bacteria as the test organisms, 151 species of British marine algae have been screened for the production of antibiotics. Of these, Asparagopsis armata, Bonnemaisonia asparagoides, Bonnemaisonia hamifera, Chondrus crispus, Dilsea carnosa, Gloiosiphonia capillaris, Sphondylothamnion multifidum, Desmarestia aculeata, Desmarestia ligulata, Laminaria digitata, Dictyopteris membranacea, Dictyota dichotoma, Halidrys siliquosa and most members of the family Rhodomelaceae appear to possess outstanding antibacterial properties. Although the production of antibiotics would appear to be a characteristic of several families, it has not been possible to establish any major correlation between taxonomy and antibiotic production. In the case of two closely related and morphologically similar species, Chondrus crispus and Gigartina stellata, the former possesses considerable degrees of antimicrobial activity whilst the latter exhibits no such activity. The results also indicate that the production of antibiotics by the algae is affected by the season of the year.