Romain Gastineau

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 micromolphotons 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.

Biological activities of purified marennine, the blue pigment responsible for the greening of oysters

Marennine, the blue pigment produced by the diatom Haslea ostrearia , exists in two different forms, the intra- and extracellular forms. We investigated the antibacterial, antiviral, and antiproliferative properties of both of these forms. Both forms of marennine inhibited the development of marine bacteria, in particular the pathogenic organism Vibrio aesturianus , at concentrations as low as 1 μg/mL, but they did not display any effect on a wide range of pathogenic bacteria that are relevant for food safety. Both forms of the pigment produced by H. ostrearia also exhibited antiviral activity against the HSV1 herpes virus, with intra- and extracellular marennine having EC(50) values of 24.0 and 27.0 μg/mL, respectively. These values are 2 orders of magnitude higher than the value for the reference drug, Zovirax. Moreover, both forms of marennine were effective in slowing or inhibiting the proliferation of cancer cells. This study confirms the potential of marennine as a biologically active organic molecule, which could have a protective effect on bivalves, which filter seawater and fix the pigment on their gills. Moreover, marennine could be used in food engineering and chemistry as a natural blue pigment. However, despite that it is eaten and possibly assimilated by green oyster consumers, it also deserves in depth evaluation before being considered for use as a nutraceutical.

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.

A (2)H solid-state NMR study of the effect of antimicrobial agents on intact Escherichia coli without mutating.

Solid-state nuclear magnetic resonance (NMR) is a useful tool to probe the organization and dynamics of phospholipids in bilayers. The interactions of molecules with membranes are usually studied with model systems; however, the complex composition of biological membranes motivates such investigations on intact cells. We have thus developed a protocol to deuterate membrane phospholipids in Escherichia coli without mutating to facilitate (2)H solid-state NMR studies on intact bacteria. By exploiting the natural lipid biosynthesis pathway and using perdeuterated palmitic acid, our results show that 76% deuteration of the phospholipid fatty acid chains was attained. To verify the responsiveness of these membrane-deuterated E. coli, the effect of known antimicrobial agents was studied. (2)H solid-state NMR spectra combined to spectral moment analysis support the insertion of the antibiotic polymyxin B lipid tail in the bacterial membrane. The use of membrane-deuterated bacteria was shown to be important in cases where antibiotic action of molecules relies on the interaction with lipopolysaccharides. This is the case of fullerenol nanoparticles which showed a different effect on intact cells when compared to dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol membranes. Our results also suggest that membrane rigidification could play a role in the biocide activity of the detergent cetyltrimethyammonium chloride. Finally, the deuterated E. coli were used to verify the potential antibacterial effect of a marennine-like pigment produced by marine microalgae. We were able to detect a different perturbation of the bacteria membranes by intra- and extracellular forms of the pigment, thus providing valuable information on their action mechanism and suggesting structural differences.

Marennine, Promising Blue Pigments from a Widespread Haslea Diatom Species Complex

In diatoms, the main photosynthetic pigments are chlorophylls a and c, fucoxanthin, diadinoxanthin and diatoxanthin. The marine pennate diatom Haslea ostrearia has long been known for producing, in addition to these generic pigments, a water-soluble blue pigment, marennine. This pigment, responsible for the greening of oysters in western France, presents different biological activities: allelopathic, antioxidant, antibacterial, antiviral, and growth-inhibiting. A method to extract and purify marennine has been developed, but its chemical structure could hitherto not be resolved. For decades, H. ostrearia was the only organism known to produce marennine, and can be found worldwide. Our knowledge about H. ostrearia-like diatom biodiversity has recently been extended with the discovery of several new species of blue diatoms, the recently described H. karadagensis, H. silbo sp. inedit. and H. provincialis sp. inedit. These blue diatoms produce different marennine-like pigments, which belong to the same chemical family and present similar biological activities. Aside from being a potential source of natural blue pigments, H. ostrearia-like diatoms thus present a commercial potential for aquaculture, cosmetics, food and health industries.

Antimicrobial Compounds from Eukaryotic Microalgae against Human Pathogens and Diseases in Aquaculture

The search for novel compounds of marine origin has increased in the last decades for their application in various areas such as pharmaceutical, human or animal nutrition, cosmetics or bioenergy. In this context of blue technology development, microalgae are of particular interest due to their immense biodiversity and their relatively simple growth needs. In this review, we discuss about the promising use of microalgae and microalgal compounds as sources of natural antibiotics against human pathogens but also about their potential to limit microbial infections in aquaculture. An alternative to conventional antibiotics is needed as the microbial resistance to these drugs is increasing in humans and animals. Furthermore, using natural antibiotics for livestock could meet the consumer demand to avoid chemicals in food, would support a sustainable aquaculture and present the advantage of being environmentally friendly. Using natural and renewable microalgal compounds is still in its early days, but considering the important research development and rapid improvement in culture, extraction and purification processes, the valorization of microalgae will surely extend in the future.

Haslea ostrearia-like Diatoms: Biodiversity out of the Blue

Abstract Diatoms are usually referred to as golden-brown microalgae, due to the colour of their plastids and to their pigment composition, mainly carotenoids (fucoxanthin, diadinoxanthin, diatoxanthin), which mask chlorophylls a and c . The species Haslea ostrearia Gaillon/Bory (Simonsen) appears unique because of its extraplastidial bluish colour, a consequence of the presence of a water-soluble blue pigment at cell apices, marennine. When released in seawater, marennine can be fixed on gills of oysters and other bivalves, which turn green. This greening phenomenon is economically exploited in Southwestern France, as it gives an added value to oysters. For decades, this singularity ascribed a worldwide distribution to H. ostrearia , first as Vibrio ostrearius , then Navicula ostrearia , last as H. ostrearia , when the genus Haslea was proposed by R. Simonsen (1974) . Indeed, this ‘birthmark’ (presence of blue apices) made H. ostrearia easily recognisable without further scrutiny and identification of the microalga as well as its presence easily deduced from the greening of bivalves. Consequently, the widely admitted cosmopolitan character of H. ostrearia has only been questioned recently, following the discovery in 2008, of a new species of blue diatom in the Black Sea, Haslea karadagensis . The biodiversity of blue diatoms suddenly increased with the finding of other blue species in the Mediterranean Sea, the Canary Islands, etc., the taxonomic characterization of which is in progress. This review thus focuses on the unsuspected biodiversity of blue diatoms within the genus Haslea . Methods for species determination (morphometrics, chemotaxonomy, genomics), as well as a new species, are presented and discussed.

Inheritance of mitochondrial DNA in the Pennate diatom Haslea ostrearia (Naviculaceae) during auxosporulation suggests a uniparental transmission.

We present the first study examining mtDNA transmission in diatoms, using sexual progeny of the pennate species Haslea ostrearia (Naviculaceae). A fragment of the cytochrome oxidase subunit I gene (cox1) with 7 nucleic substitutions between parental clones was used as a parental tracer in 16 F1 clones obtained from two pairs of mating crosses. Each cross involved a parental clone isolated from France (Bay of Bourgneuf) and Sweden (Kattegat Bay). We determined that all progeny possessed only one cox1 parental haplotype. These results suggest that the mitochondrial DNA transmission in H. ostrearia is uniparental. Implications and new topics of investigation are discussed.

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

Abstract 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.

Does allelopathy affect co-culturing Haslea ostrearia with other microalgae relevant to aquaculture?

Haslea ostrearia is a marine diatom known to produce marennine, a water-soluble blue-green pigment responsible for the greening of oysters in ponds along the French Atlantic coast. This phenomenon occurs seasonally when H. ostrearia blooms in oyster ponds, and it increases the economic value of cultured oysters. From an ecological perspective, H. ostrearia blooms are accompanied by a decrease in the abundance of other microalgae, suggesting that this diatom produces allelochemicals. Recent studies showed that purified marennine has other biological activities, for instance antioxidant, antibacterial, and antiviral activities, which could be used in aquaculture to promote this pigment as a natural antipathogen agent. One important issue regarding the possible use of H. ostrearia in aquaculture as a mixed algal diet, however, is the importance of marennine allelopathy. In this study, we investigated the allelopathic effect of H. ostrearia on the growth of five microalgal species relevant to aquaculture: Chaetoceros calcitrans, Skeletonema costatum, Phaeodactylum tricornutum, Tetraselmis suecica, and Tisochrysis lutea. Allelopathic tests were realized by co-culturing these microalgae with H. ostrearia in batch and in semi-continuous mode, based on initial biovolume ratios. Our findings showed that inhibition of the growth of microalgae due to the presence of H. ostrearia and marennine was species dependent. Skeletonema costatum, C. calcitrans, and T. lutea were significantly more sensitive, whereas T. suecica and P. tricornutum appeared to be more resistant. Growth irradiance significantly influenced the allelopathic effect against the sensitive species S. costatum, and the H. ostrearia production of marennine increases with irradiance. Data presented in this study partly support the hypothesis that marennine released into the culture medium possibly acts as an allelochemical compound, thus explaining the dominance of H. ostrearia and the loss of sensitive algae in oyster ponds, but also that some species are insensitive, which allows co-culturing and use in a mixed algal diet in aquaculture.