Phytoplankton distribution during two contrasted summers in a Mediterranean harbour: combining automated submersible flow cytometry with conventional techniques.

Automated in situ flow cytometry, high-pressure liquid chromatography (HPLC), optical microscopy and fluorometry were combined to monitor phytoplankton over two summer periods (2005 and 2006). In 2006, temperature was higher and nutrients lower than in 2005, generating differences in the phytoplankton assemblages (i.e., abundance and structure). Pigment-size classes based on daily HPLC analysis provided evidence for higher proportions of picoplankton and nanoplankton with higher biomass in 2005 and a dominance of microplankton with lower biomass in 2006, the latter with lower specific diversity, as evidenced by weekly microscopy analyses. Total chlorophyll a estimations from fluorometry measurements recorded every 30 min were higher in 2005 than in 2006, as for the HPLC chlorophyll a concentrations. An automated in situ flow cytometer (Thyssen et al., J Plankton Res 30(9):1027-1040, 2008a) sampled seawater every 30 min. Data analysis yielded the resolution of seven clusters based on light scatter and fluorescence. In 2006, an increase in abundance of the largest cells was observed, confirming pigment and microscopy data. The results suggest that the ecosystem was on a constant renewing process in summer 2005 due to a strong wind event and on a highly productive and recycling way in summer 2006 due to stratification of the upper water layer. Automated submersible flow cytometry confirms to be a powerful tool providing high-resolution data by monitoring phytoplankton at the single cell level. This technology gives access to the shape of the light scatter and fluorescence signals generated by each cell passing through a laser beam and that are linked to size, structure and pigment content of the target cell. When combined with conventional techniques, it further improves our understanding of phytoplankton assemblages.

Comparative study of three analysis methods (TTGE, flow cytometry and HPLC) for xenobiotic impact assessment on phytoplankton communities.

The impacts of the fungicide Opus (epoxiconazole) on marine phytoplankton communities were assessed in a 12-day field experiment using in situ microcosms maintained underwater at 6 m depth. Three community analysis methods were compared for their sensitivity threshold in fungicide impact detection. When phytoplankton communities were exposed to 1 microg l(-1) of epoxiconazole, no effects could be demonstrated using TTGE (Temporal Temperature Gradient gel Electrophoresis), flow cytometry or HPLC. When exposed to 10 microg l(-1), TTGE fingerprints from PCR amplified 18S rDNA of communities exhibited significant differences compared with controls (ANOSIM, P = 0.028). Neither flow cytometry counts, nor HPLC pigment profiles allowed to show significant differences in microcosms exposed to 10 microg l(-1) of epoxiconazole. When exposed to 100 microg l(-1), all three methods allowed to detect significant differences in treated microcosms, as compared to controls. The TTGE analysis appears in this study as the most sensitive method for fungicide impact assessment on eukaryote microbial communities.

Regiospecific oxygenation of alkenones in the benthic haptophyte Chrysotila lamellosa Anand HAP 17.

Two groups of previously unidentified C37-C39 epoxyalkenones and alkenediones were detected in late stationary phase cultures of the haptophyte microalga Chrysotila lamellosa. The formation of these compounds is attributed to the involvement of enzymatic processes acting specifically on the C-21 or C-22 allylic carbon and the omega15 double bond of methyl and ethyl alkenones respectively. Thus, the epoxyalkenones appear to be derivatives of alkenones where the omega15 double bond is oxidized to the epoxide. These epoxyalkenones disappear as the cells age to be replaced by a series of alkenediones. The structures of these compounds indicate that they are derivatives of methyl and ethyl alkenones with an additional carbonyl group on the C-21 or C-22 carbon respectively and without the omega15 double bond. We propose that these compounds are formed by an initial regiospecific lipoxygenase-catalyzed peroxidation of methyl and ethyl alkenones on their C-21 or C-22 allylic carbon, respectively. Lipohydroperoxidase-catalyzed homolytic cleavage of the O-O bond could then result in the formation of conjugated ketones which may then undergo a saturation reaction to form the diketones identified. This work demonstrates that alkenones can be degraded by enzymatic reactions in senescent cells, and by implication this could also occur in the natural environment.

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).

Long-chain alkenones and related compounds in the benthic haptophyte Chrysotila lamellosa Anand HAP 17.

The neutral lipid compositions of the coastal haptophyte Chrysotila lamellosa HAP 17 grown in batch culture at 10 and 20 degrees C have been determined. A comparison was also made between the lipid compositions of cells harvested in early and late stationary phase. This species contains a suite of very long-chain C(37)-C(40) alkenones and alkenoates as found in a few microalgae from the Haptophyta. The distributions of these compounds show some differences to earlier reports of different strains of this alga, which are only in part attributable to culture conditions. A suite of long-chain alkenols, the reduced form of the alkenones, was characterized for the first time. The abundance of these compounds was only 1.5% of that of the corresponding alkenones, and the relative proportion of C(37)-C(38) constituents depended on growth temperature. These data show that haptophyte algae are a possible source of the alkenols found in some marine sediments, but the small amounts found suggest that other sources such as bacterial reduction of alkenones are more likely in highly reducing sediments. A mixture of C(29)-C(33) n-alkenes, dominated by the C(31:1) monoene, was found in marked contrast to previous analyses of other strains which reported only the presence of a C(31:2) diene. The sterol distribution included the common haptophyte sterol 24alpha-methylcholesta-5,22E-dien-3beta-ol (epi-brassicasterol) as well as significant amounts of Delta(5)- and Delta(5,22)-C(29) sterols.

On the origin of cis-vaccenic acid photodegradation products in the marine environment.

The origin of 11-hydroxyoctadec-trans-12-enoic and 12-hydroxyoctadec-trans-10-enoic acids (photodegradation products of cis-vaccenic acid) in the marine environment was investigated. cis-Vaccenic acid is commonly used as a bacterial biomarker; however, in heterotrophic bacteria the observed rates of cis-vaccenic acid photodegradation are negligible. Here, two hypotheses explaining the source of the photoproducts were tested. According to the first hypothesis, the photoproducts originate from aerobic anoxygenic bacteria, i.e., photoheterotrophic organisms using bacteriochlorophyll-containing reaction centers. Alternatively, the photoproducts come from a heterotrophic bacterial community closely associated with senescent phytoplanktonic cells. cis-Vaccenic acid photodegradation was detected in both experimental setups. However, a detailed comparison of the cis-vaccenic acid photodegradation patterns with those observed in particulate matter samples of the DYFAMED station (Mediterranean Sea) suggests that photodegradation of heterotrophic bacteria attached to senescent phytoplanktonic cells constitutes the more likely source of cis-vaccenic acid oxidation products detected in situ.

A flow cytometric approach to assess phytoplankton respiration.

Microbial respiration in the ocean is considered as the major process representative of the organic matter biological oxidation. The corresponding metabolic CO2 production was estimated to be about 22 Pg C y(-1). However, the in situ respiration rate is generally too low (by several orders of magnitude) to be accessible to the available direct measurement methods. Some fluorescent probes, such as DiOC6(3) (Molecular Probes, USA) have been shown to be very sensitive to changes in the proton electrochemical potential difference (DeltamuH+), characterising mitochondrial and plasmic membranes bearing the cell respiratory system in eukaryotic and prokaryotic cells respectively. In mitochondria, DeltamuH+ is linked to the flux of oxygen uptake by a linear relationship. To our knowledge, no such relationship has been established in the case of whole marine cells. In the present work, we addressed the dark respiration rate of the Chlorophyceae Dunaliella tertiolecta (Butcher) in axenic cultures, both directly by using a highly sensitive oxygraph (Oroboros) and by staining cells with DiOC6(3). We found and standardized a linear relationship between oxygen uptake by D. tertiolecta and its green fluorescence induced by DiOC6(3), enabling the determination by flow cytometry of the respiration rate of D. tertiolecta.