Variable stereochemistry in highly branched isoprenoids from diatoms

C(25) highly branched isoprenoid (HBI) alkenes are ubiquitous lipids found in geochemical samples around the globe. The origins of these widespread geochemicals are believed to be restricted to a limited number of diatoms, including Haslea ostrearia (and related species), Rhizosolenia setigera, and Pleurosigma intermedium. The unsaturation of the HBI alkenes ranges from 2-6 in different species and cultures. The number of stereogenic centres is usually limited to two in the HBI alkenes due to double bond positions. The relative and/or absolute configurations for these have been determined for a range of HBI alkenes produced from different diatoms cultured under a number of growth conditions. These determinations have involved a combined spectroscopic and chromatographic analysis using NMR spectroscopy and chiral gas chromatography, respectively. HBIs isolated from Haslea spp. belong to a specific structural type which exhibit configurational diastereoisomerism, while those isolated from P. intermedium and R. setigera represent a different structural type and usually exist as mixtures of geometric isomers only. HBIs are reported from a new species of diatom whose stereochemical properties lie between those found for Haslea spp. and P. intermedium.     

Configurations of polyunsaturated sesterterpenoids from the diatom, Haslea ostrearia

The partial configurations of C25 isoprenoid alkenes isolated from the diatom Haslea ostrearia Gaillon (Simonsen) have been established. A combination of NMR spectroscopy studies of the alkenes with chiral shift reagents in conjunction with soluble silver beta-diketonate complexes and enantioselective gas chromatography of oxidation products of the alkenes was used. Unexpected differences in highly branched isoprenoid isomer configurations were observed between different laboratory cultures of the alga.     

Haslea salstonica sp. nov. and Haslea pseudostrearia sp. nov. (Bacillariophyta), two new epibenthic diatoms from the Kingsbridge estuary, United Kingdom

Two new diatom species, Haslea salstonica and Haslea pseudostrearia are described in light and electron microscopy and compared with two well-known members of Haslea. Scanning electron microscope observations confirm that the new species belong to the genus Haslea. This study extends previous observations on the genus, particularly with respect to the development of a pseudostauros. The characteristic features of the genus are discussed briefly.     

Artificial cell-immobilization: a model simulating immobilization in natural environments?

A study of the marine diatom Haslea ostrearia was performed in controlled culture conditions. Biofilm formation by cells grown in liquid medium was studied through macroscopic events and extracellular polysaccharides (EPS) production. Alcian blue staining and image analysis methods were used to quantify EPS production in regard to the usual pattern (lag, exponential and stationary phases) of microalgal growth. Simultaneously, an artificial immobilization of H. ostrearia cells was carried out using an agar layer. Samples from liquid medium and artificially immobilized cultures were studied in scanning electron microscopy. They revealed the structure of the entrapping material and progressive changes of the natural biofilm along culture ageing. The use of artificially immobilized cultures for solute diffusivity investigations is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

C(25) highly branched isoprenoid alkenes from the marine benthic diatom Pleurosigma strigosum

The hydrocarbon composition of the marine diatom Pleurosigma strigosum isolated from coastal Mediterranean sediments is described. A suite of five C(25) highly branched isoprenoid (HBI) alkenes with 2-5 double bonds were detected together with n-C(21:4) and n-C(21:5) alkenes and squalene. The analysis by (1)H and (13)C NMR spectroscopy of two isolated HBI alkenes allowed the structural identification of a novel C(25) HBI triene (2,6,10,14-tetramethyl-7-(3-methylpent-4-enyl)-pentadeca-5E,13-diene) and the first identification in diatom cells of 2,6,10,14-tetramethyl-7-(3-methylpent-4-enyl)-pentadec-5E-ene, an HBI previously detected in marine sediments and particulate matter. The other minor C(25) HBIs detected were a tetraene and a pentaene that have been previously identified in other diatoms from the genera Haslea and Rhizosolenia, and one other C(25) tetraene that could not be structurally identified. The structures of the HBI alkenes of P. strigosum were compared with those of C(25) homologues previously identified in three other Pleurosigma sp. (Pleurosigma intermedium, Pleurosigma planktonicum and Pleurosigma sp.). Unlike most structures previously reported, none of the HBI alkenes produced by P. strigosum showed an unsaturation at C7-C20, or E/Z isomerism of the trisubstituted double bond at C9-C10 (whenever present).     

A new photobioreactor for continuous marennin production with a marine diatom: influence of the light intensity and the immobilised-cell matrix (alginate beads or agar layer)

In oyster ponds, the marine diatom Haslea ostrearia synthesises and excretes a hydrosoluble pigment of commercial interest called marennin. During the benthic stage, when algal cells are naturally immobilised in their own polysaccharides, marennin production is higher. To optimise this production, axenic cultures of H. ostrearia were immobilised in a polysaccharidic matrix (alginate or agar) and introduced into a new photobioreactor device for continuous marennin production. Solute diffusion was improved using an alginate beads monolayer, leading to higher levels of cell growth (a 2-fold higher cell concentration) and marennin productivity (7.57-8.80 mg day(-1) l(-1)). An increase in the light intensity (from 3.0 to 8.5x10(16) quanta cm(-2) s(-1)) led to an earlier and 1.3-fold higher production of marennin. However, the higher light intensity led to a higher rate of cell death [0.29 instead of 0.40 ng chlorophyll a (10(6) cells)(-1)]. Due to the secondary nature of marennin metabolism, it would be necessary to alternate between culture conditions favouring cell growth (moderate light intensity and no limiting nitrate supply) and those promoting marennin production (high light intensity and limiting nitrate supply).     

STEROLIC BIOMARKERS IN MARINE PHYTOPLANKTON. II. FREE AND CONJUGATED STEROLS OF SEVEN SPECIES USED IN MARICULTURE

The sterols of seven unicellular algae widely used in mariculture and belonging to different classes (Prasinophyceae, Haptophyceae, Eustigmatophyceae, and Diatomophyceae) were examined for free and combined forms. Under standard culture conditions, all synthesized free sterols and combined forms (steryl esters, acyl steryl glycosides, and steryl glycosides). Free sterols were dominant in five of the species. In contrast, sterols were mostly esterified in the eustigmatophyte Nannochloropsis oculata (Droop) Hibberd and the diatom Thalassiosira pseudonana Hasle et Heimdal, whereas in another diatom, Chaetoceros calcitrans (Paulsen) Takano, glycosylated forms represented over 60% of total sterols. The pennate diatom Haslea ostrearia (Gaillon) Simonsen synthesized large amounts of steryl glycosides consisting mainly of an unusual sterol, 23,24-dimethylcholest-5-en-3β-ol, occurring in some dinoflagellates.     

Factors influencing the distributions of polyunsaturated terpenoids in the diatom, Rhizosolenia setigera.

Polyunsaturated highly branched isoprenoid (HBI) hydrocarbon distributions of laboratory cultures of five strains of the planktonic diatom Rhizosolenia setigera (Brightwell) are shown herein to be highly variable. Some strains produced both haslenes with from three to five double bonds and rhizenes. The haslenes comprised not only Delta5 alkenes but also those with C7(20) unsaturation, including hasla-7(20),9E,Z, 23-trienes and hasla-7(20),9E,Z-13, 23-tetraenes. The rhizenes contained C7(25) unsaturation and the vinyl moiety common to all algal haslenes so far characterised. The effects of temperature and salinity on HBI composition, along with isotopic content, were determined in strain CS 389/A. Increase in growth temperature from 18 to 25 degrees C increased the degree of unsaturation in the haslenes and E to Z isomerisation in the triene. There was also an increase in unsaturation in the rhizenes at the highest growth temperature, with hexaenes dominant over the pentaenes but in the rhizenes, Z to E isomerisation increased. Increased salinity from 15 to 35 psu increased cell growth and rhizene production but decreased haslene production. Unsaturation in haslenes was not changed by increased salinity but unsaturation in the rhizenes decreased. These may reflect growth rate differences. The carbon isotopic compositions of the haslenes and rhizenes were similar to that of the major sterol at 18 degrees C, but the major HBI isomers were 3-4 per mil depleted relative to phytol released by saponification from chlorophyll a. This suggests biosynthesis of HBIs from a different isotopic pool of isopentenyl biphosphate to that from which phytol is biosynthesised. At 25 degrees C, further isotopic differences were observed. The variables controlling HBI distributions in R. setigera are still not fully understood and rationalisation of the environmental controls on the sedimentary distributions of the HBIs from R. setigera may only be possible once such factors are established.     

Light is a key factor in triggering sexual reproduction in the pennate diatom Haslea ostrearia

Sexual reproduction is an obligatory phase in the life cycle of most diatoms, as cell size decreases with successive vegetative divisions and the maximal cell size is only restored by a specialized cell, the auxospore, which follows zygote formation as a result of sexual reproduction. While in pennate diatoms the induction of sexual reproduction depends primarily on cell–cell interactions, the importance of different external factors for the induction of sexual reproduction is less well known. Here, we investigated the effects of light on sexualization in the marine benthic pennate diatom Haslea ostrearia (Gaillon) R. Simonsen. Compatible clones were crossed and exposed to different combinations of light levels, qualities, and photoperiods. Light was found to be a key factor for sexualization, and to a certain extent, to control auxosporulation in H. ostrearia . The light conditions most favorable for sexual reproduction were low irradiances (<50 μmolphotons m⁻² s⁻¹) and short photoperiods (6–10 h), conditions that prevail during winter, and to a lesser extent, the higher irradiances and longer photoperiods that correspond to the spring and fall, when blooms of this organism form in the natural environment. Auxospore formation was very rare in continuous light, and maximum in presence of red radiation, while it was never observed in darkness or in radiation other than red.     

Effects of temperature on polyunsaturation in cytostatic lipids of Haslea ostrearia

Unusual chemicals produced by the-'blue oyster' diatom, Haslea ostrearia, include the water-soluble blue pigment marennine and numerous polyunsaturated sesterterpene oils or haslenes. Aqueous extracts of the alga exhibit in vitro and in vivo activities against human lung cancer cells and anti-HIV effects. Here we report that three haslenes also demonstrate in vitro cytostatic action against a human lung cancer cell line. The most active haslene is the most unsaturated and unsaturation in the haslenes increases with increasing algal growth temperature.     

Alginate-entrapped Haslea ostrearia as inoculum for the greening of oysters

Entrapment in calcium alginate beads of the marine diatom, Haslea ostrearia, was successfully used for stock-culture managment and afterwards the sowing of ponds for the greening of oysters. After storage during almost 2 months, viable and cultivable cells were recovered from beads by dissolving alginate matrix but an original way lies in directly introducing beads in ponds and promoting natural cell leakage. © Rapid Science Ltd. 1998     

Haslea karadagensis (Bacillariophyta): a second blue diatom,recorded from the Black Sea and producing a novel blue pigment

A new species of raphid pennate diatom producing a blue pigment, Haslea karadagensis Davidovich, Gastineau & Mouget, sp. nov., was recently isolated from the Crimean coast of the Black Sea (Ukraine). This organism is very similar to the well-known Haslea ostrearia, the first described ‘blue’ diatom, which produces marennine, the pigment involved in the greening of oysters. The Ukrainian diatom, H. karadagensis, differs slightly from H. ostrearia in the structure of its frustule, and the two organisms are unable to interbreed. Two molecular markers, rbcL and the ITS1–5.8S–ITS2 sequences, showed 2% and >50% differences, respectively, between the two species. UV-visible spectrophotometry and in vivo confocal micro-Raman spectroscopy were used to compare the pigment of H. karadagensis with marennine. Both pigments showed absorption bands in the UV and red regions, but the positions of the maxima differ between the pigments. Significant differences were observed by micro-Raman spectroscopy in the 1000–1700?cm^?1 wavenumber range, revealing that the pigments are different molecules. Haslea karadagensis is the first example of a new ‘blue’ diatom and produces a novel blue pigment.     

Comparison of cryopreservation methods for the long term storage of the marine diatom Haslea ostrearia (simonsen)

Long term maintenance of microalgal strains by serial subculturing is often expensive and time-consuming. Alternative methods, such as cryopreservation, present several benefits and thus seem more relevant. Our study aimed at comparing two cryopreservation procedures applied to the marine diatom Haslea ostrearia (Simonsen): (1) a two-step freezing method in liquid media using 5%, 10% and 20% MeOH, Me₂SO or Glycerol, and (2) an immobilization-dehydration method consisting in an algal cell entrapped in 0.7 M sucrose dehydrated and air-flow desiccated calcium alginate beads before “direct” or “two-step” freezing. Our results showed that the cryopreservation of H. ostrearia was feasible. With the two-step freezing protocol only Me₂SO maintained cell viability without contamination but the low percentage of viability (<10%) prevents its use. Conversely, the immobilization-dehydration methods tested in this study were effective. Average viability of 57% and 77% were obtained with the “direct” and the “two step” cooling assays respectively, ensuring preservation of the genetic traits of H. ostrearia.     

A new blue-pigmented hasleoid diatom,Haslea provincialis,from the Mediterranean Sea

Haslea provincialis Gastineau, Hansen & Mouget, sp. nov., is a new, morphologically semicryptic blue diatom discovered on the French shores of the Mediterranean Sea. Like H. ostrearia and H. karadagensis, H. provincialis shares the capacity to synthesize a marennine-like blue pigment. Sexual reproduction between clones of H. provincialis has been repeatedly observed and resulted in viable initial cells. There were no sexual interactions with sexually competent clones of H. ostrearia or H. karadagensis, as would be expected for a separate biological species. There are strong similarities between the H. provincialis pigment and the marennine produced by H. ostrearia, evidenced by UV-visible spectrophotometry and Raman spectrometry. However, unlike the marennine from H. ostrearia, no differences were found between the extracellular and the intracellular forms of the pigment in H. provincialis. This indicates that the synthesis pathways and excretion mechanisms among the three ‘blue’ Haslea may be species-specific. Molecular taxonomy and phylogeny (based on rbcL, cox1 and SSU V4 DNA sequences) confirmed the distinct position of this species among the blue Haslea species. Haslea provincialis occurs in environments from which H. ostrearia has already been reported (mostly based on the presence of the blue cell vacuoles). Possible species misidentifications and the impact of the complex geological history of the Mediterranean Sea on blue diatom diversification are also discussed.     

Phytotoxicity and ultrastructural effects of gymnopusin from the orchid Maxillaria densa on duckweed (Lemna pausicostata) frond and root tissues

The effect of life cycle on the distributions of C(25) and C(30) highly branched isoprenoid (HBI) alkene lipids has been investigated for the marine diatom Rhizosolenia setigera. The concentrations of the C(30) compounds are largely independent of the cell volume, though the ratios of the individual isomers possessing five and six double bonds show a dependence on the position of the cell during its life cycle, especially during auxosporulation. In contrast to the C(30) pseudo-homologues, the C(25) isomers are not always detected in cultures of R. setigera. The biosynthesis of the C(25) HBIs would appear to result from the onset of auxosporulation, with further changes to their distributions taking place after this phase, including the formation of more unsaturated isomers. The results of this investigation may be used in part to explain the large variations in these lipids reported previously.     

Flow cytometric determination of zoospore DNA content and population DNA distribution in cultured Pfiesteria spp. (Pyrrhophyta)

The relative cellular DNA content from 23 different clonal cultures of Pfiesteria spp. zoospores was determined using a DNA fluorochrome and flow cytometry. Significant differences between Pfiesteria piscicida and P. shumwayae were detected, both in mean zoospore DNA content and population cell cycle DNA distribution. Intraspecific differences in DNA content were found between clonal zoospore cultures established from different geographical regions. Long-term cultures (years) of P. piscicida were available for testing, and a negative correlation was observed between zoospore DNA content and time in culture. Zoospore cell cycle-related DNA distributions were also markedly different between the two species in these clonal cultures. In most cultures tested, P. piscicida zoospores exhibited bimodal DNA flow histograms with G1-S-G2+M distributions, typical of eukaryotic asynchronously cycling cells. In contrast, cultures of P. shumwayae zoospores exhibited one DNA peak distribution, indicative of synchronized cells. The data are consistent with the hypothesis that P. shumwayae zoospores are interphasic cells, and mitosis in zoospore cultures of this species predominantly occurs as benthic or adherent non-motile division cysts. Light microscopy observations of the nuclear condition of electrostatically sorted zoospores of each Pfiesteria species also support this hypothesis. If highly conserved, this disparity in modes of vegetative reproduction would ramify the population dynamics of the two Pfiesteria species.     

Morphology and development of secretory structures in Hypericum perforatum and H. richeri

Secretory organs are a specialised anatomical feature of plants, these tissues function in the production and/or storage of specific chemical substances, which often have pharmacological properties. The genus Hypericum is characterised by the presence of specialised secretory structures such as black nodules, translucent glands and secretory canals. The presence of these structures is not homogeneous for all the species or between the various floral and vegetative parts of the plants. In this study, we have compared the distributions, morphology and development of secretory structures in the leaves of H. perforatum and H. richeri. Whilst, black nodules occurred in both species, translucent glands followed different development stages depending on the leaf age.     

The Effect of Temperature on Vegetative Growth and Sexual Reproduction of Two Diatoms from the Genus <Emphasis Type="Italic">Haslea</Emphasis> Simonsen

The effect of temperature on sexual reproduction and the rate of vegetative division were investigated in laboratory experiments with Haslea ostrearia (Gaillon) Simonsen from the Atlantic and H. karadagensis Davidovich, Gastineau et Mouget from the Black Sea. In the range of 10–26°С, a temperature increase was favorable for vegetative growth; the maximum growth rate was recorded at the highest temperature of the range. In contrast to vegetative growth, sexual reproduction of both algal species was effective in the temperature range of 10–18°С, but did not occur at temperatures of 23°С. The optimal temperature for auxosporulation was in H. karadagensis somewhat lower than in H. ostrearia. The determined physiological optimums for sexual reproduction can explain the cell-size distribution that is characteristic of the natural population of H. karadagensis in different seasons of the year.     

Epipelic diatoms blooming in Isahaya Tidal Flat in the Ariake Sea, Japan, before the drainage following the Isahaya-Bay Reclamation Project

A blooming diatom sample from Isahaya Tidal Flat in the Ariake Sea, south‐west Japan, before the bay was closed and drained following the Isahaya‐Bay Reclamation Project, is taxonomically examined. When the sample was collected on 25 May 1996, the area was a muddy tidal flat. Altogether, 103 diatom species were observed within 1000 counts of frustules. Both the dominant and subdominant diatom species were large raphid species. Light microscopy and scanning electron microscopy observation revealed that the dominant species was Haslea nipkowii (F.Meister) Poulin et G. Masse, which was originally described as a species of Gymsigma. So far, a report of correctly identified H. nipkowii has been presented only from China, because H. nipkowii sensu Poulin et al. from USA and France probably belongs to the other taxon, Gymsigma pallidum Riznyk. The subdominant species is described as a new species, Nitzschia gyrosigmasp. nov. This new species is similar to Nitzschia sigmoidea under light microscopy, but scanning electron microscopy observation reveals that their fine structures are quite different. Nitzschia granulata var. hyalina is transferred to the genus of Tryblionella and raised to an independent species, Tryblionella hyalina (Amosse) comb. nov.