Extraction and quantitative analysis of the blue-green pigment “marennine” synthesized by the diatom Haslea ostrearia

This study reports further information on mareninne, awater-soluble blue-green pigment synthesized by the diatom Hasleaostrearia, which is essential to the greening of maturing oysters inFrench production areas. The extraction process is reported, as well aspreliminary characterization of a partially purified marennine extract,including quantitative spectrophotometric analysis of intracellular pigment. Amean specific extinction coefficient, E ^1% _1cm = 17.2 at 669 nm, is proposed. Results forquantitative determination of marennine accumulated in cells during batchcultures are presented, and their importance for pigment production isconsidered in relation to potential industrial applications.     

Purification of the blue-green pigment “marennine” from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen

The diatom Haslea ostrearia that lives in oyster ponds has the distinctive feature of synthesizing “marennine”, a blue-green pigment of which the chemical nature still remains unknown. This pigment is responsible for the greening of oyster gills. Here, we report a new method for extraction and purification of intracellular (accumulated in the apex of the cell) and extracellular (released into the external medium) forms of the pigment. Intracellular marennine is obtained by extraction from blue algal pellets with a carbonate buffer. The extract is then centrifuged and filtered. Extracellular marennine is obtained by clarification of blue-coloured culture medium. Both extracts are then purified by a semi-preparative process, using ultrafiltration through membranes and anion-exchange chromatography. This procedure allows us to produce native pigment displaying the degree of purity required to enter upon the molecular characterisation of marennine. By this process, about 35% of the initial amount of pigment can be recovered. If necessary, this method could be easily scaled up to a larger production system to accommodate potential industrial applications.     

VARIATION IN PATTERNS OF VALVE MORPHOGENESIS BETWEEN REPRESENTATIVES OF SIX BIRAPHID DIATOM GENERA (BACILLARIOPHYCEAE)

Scanning electron microscopic studies of silica valve formation in naviculoid diatoms representing six different genera revealed that the precise sequence of depositional events varied among genera. Valve deposition begins with the formation of the raphe sternum, from which virgae (lateral outgrowths) extend. Areolae (pores) are formed between the virgae by the fusion of cross-extensions (vimines). In most of the species studied ( Craticula ambigua (Kützing) D. G. Mann, Frustulia vulgaris (Thwaites) De Toni, Craspedostauros australis E. J. Cox, and Gomphonema truncatum Ehrenberg), areola (pore) formation began near the raphe sternum before completion of the valve margin, but in Pinnularia gibba Ehrenberg the valve margin fused before the areolae were formed. Silica deposition in all these taxa was mainly distal to proximal (with respect to the cytoplasm), but in Haslea sp. it was mainly proximal to distal. Haslea also differed in that areolae were defined as the valve margin was completed. These data have also contributed to the interpretation of taxonomically important features, such as raphe endings. In P. gibba the internal central raphe fissures were laterally deflected but subsequently obscured by additional silicification of the valve, whereas in G. truncatum they were initially straight, becoming laterally deflected as valves mature. External raphe fissures in Frustulia became Y-shaped only just before maturity; in immature valves they were dotlike, as in Amphipleura Kützing. The comparison of developmental pathways in diatoms is a useful adjunct to morphological and other approaches in diatom systematics and warrants renewed attention.     

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.     

Microalgal cell immobilization for the long-term storage of the marine diatom Haslea ostrearia

To preserve the characteristics of the marine diatom Haslea ostrearia during long term storage, particularly size and shape, the algal cells were immobilized in alginate beads and stored at 4 ^∘C at reduced irradiance up to 4 months. Two clones of different size (Ho34, 63 μm and Ho40, 78 μm) were studied. With Ho34, a 10.4% decrease of the size was shown after 120 days, by using the conventional storage management, while it did not exceed 2.2% with immobilized cells. Consequently, H. ostrearia would have auxosporulated after 9 months compared to 52 months. At the same time, the rate of distortion (aberrant valve structure) free Ho34 cells reached 86% while no distorted immobilized cells were observed. Chorophyll content in cells showed that all the cells were alive up to 60 days and after this time cells immobilized in the core of the beads most probably suffered from the poor light diffusion. Culturability of the immobilized cells was tested immediately after their immobilization and after 60 and 120 days of storage. The delay (at least 5) before immobilized cells released from the beads decreased with the time of storage, because of the embrittlement of the beads during the storage. Once in fresh medium, the cells actively multiplied. We concluded that immobilization strongly slowed down the decrease in frustule size with time and allowed the storage of concentrated and calibrated inocula which could be inoculated directly in liquid culture medium without needing to dissolve the beads.     

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.     

Tetrasporangia in Asparagopsis armata

The first record for tetrasporangia in Asparagopsis armata Harv. (Falkenbergia rufolanosa (Harv.) Schm.) is given for the British Isles.     

Preliminary characterisation of the blue-green pigment “marennine” from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen

Haslea ostrearia is a common marine tychopelagic diatom which has the particularity of synthesizing a blue-green hydrosoluble pigment called “marennine”. This pigment, when released into the external medium, is known to be responsible for the colour of oyster gills. Here we present results for main biophysical and biochemical characteristics of pure intra- and extracellular marennine. Tests for chemical determination show that the nature of the two forms of marennine cannot be distinguished and could be related to a polyphenolic compound. Nevertheless, based on spectral properties and the molecular weight, which is about 10751 ± 1 and 9893 ± 1 Da, for the intracellular and extracellular forms respectively, we assess that the pigment accumulated in the apex of the cell and the one released in the external medium have probably distinct molecular structures.     

A simple procedure for polymerase chain reaction of the PSBA gene in algae: application to the screening of mutations conferring atrazine resistance and discrimination of natural populations of Porphyra linearis

A simplified procedure is described for polymerase chain reaction (PCR) of a partial sequence (bp 601–893) of the plastid gene psbA in the rhodophyte Porphyra linearis and the diatoms Haslea ostreria and Skeletonema costatum. This procedure involves the use of all tissues of P. linearis and live cell suspensions of H. ostreria or S. costatum as DNA templates, without any further purification of DNA. As in the case of PCR with DNA extracts, a single major band of the expected size (292 bp) was obtained after PCR for the three species. Sequences of the amplified fragments were aligned, confirming that the amplified products were part of the psbA gene. The method was then used to screen mutations in partial psbA genes of 23 samples of P. linearis collected at four different stations along the mid-Atlantic coast of France. An alignment was obtained indicating the existence of mutations, though not in codons known for herbicide resistance. All mutations found were silent. However, genetic polymorphism discriminated between samples collected from two stations. The method employed allows rapid amplification of the herbicide target gene and simplifies the procedure for screening mutations or populations in algae. Its application to other genes and species is considered.