Patrick Gentien

In-situ depth profiling of particle sizes

A new in-situ particle-size profiling system is presented. It allows direct determination of particle distribution spectra in 30 size classes ranging from 0.7 to 400 μm-equivalent diameter as well as an unbiased estimation of the total particle load. This profiler includes among standard probes a particle-size analyser. This new probe uses the well-known principle of diffraction pattern analysis previously used in bench-top instruments. However, the rigorous mechanical tolerances imposed by oceanographic use required a completely new design. Its description and validation is presented as well as some oceanographic applications. This instrument presents numerous advantages in oceanographic research. Its use in different European waters has demonstrated its reliability and allowed the description of common features of the profiles, particularly the accumulation of aggregates or mucilages and, in some cases, the confinement of dinoflagellates at the pycnocline. This makes possible a new sampling strategy for toxic dinoflagellates and improvements in the study of sedimentation and flocculation processes.

Motility and autotoxicity in Karenia mikimotoi (Dinophyceae)

Karenia mikimotoi is one of the most common red-tide dinoflagellates proliferating in the eastern North Atlantic and around Japan. Kills of marine fauna are associated with its blooms. In mixed water columns it migrates vertically, while in stratified water columns, the population remains confined within pycnocline layers. Wind events, increasing mixing and agitation initiate declines in its populations. This paper is focused on the formulation of mortality rate relative to shear rate. Autotoxicity is demonstrated by the use of a synthetic toxin. Bioconvection observed in cultures allows the establishment of a trade-off between phototropism, which leads to the local accumulation of cells, and their autotoxicity, which would prevent cell concentration. The combination of these processes allows diffusion of the toxin into the underlying water, where it subsequently degrades. Confinement of the population in the pycnocline layer results also from another trade-off between growth conditions and shear-rate-modulated mortality. A simplified encounter kernel was introduced into the population dynamics equation to account for a mortality factor. Under realistic forcing conditions with a small number of parameters, this model reproduced the confinement of the population in the pycnocline layer, the proper timing and the duration of the recurrent K. mikimotoi bloom on the Ushant front (France).

Haemolytic glycoglycerolipids from Gymnodinium species

Glycoglycerolipids derived from microalgae can be a source of biologically active substances including toxins. Such glycolipids were analysed in two isolates of toxic marine dinoflagellates from European waters. The lipids of Gymnodinium mikimotoi contained 17% of monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), while in Gymnodinium sp. the proportion was 35%. MGDG and DGDG from both species were haemolytic. The major unsaturated fatty acid in both algal glycolipids was 18:5 omega 3.

Rapid enzyme-linked immunosorbent assay for detection of the algal toxin domoic acid

Abstract Domoic acid (DA) is a potent toxin produced by bloom-forming phytoplankton in the genus Pseudo-nitzschia, which is responsible for causing amnesic shellfish poisoning (ASP) in humans. ASP symptoms include vomiting, diarrhea, and in more severe cases confusion, loss of memory, disorientation, and even coma or death. This paper describes the development and validation of a rapid, sensitive, enzyme linked immunosorbent assay test kit for detecting DA using a monoclonal antibody. The assay gives equivalent results to those obtained using standard high performance liquid chromatography, fluorenylmethoxycarbonyl high performance liquid chromatography, or liquid chromatography—mass spectrometry methods. It has a linear range from 0.1–3 ppb and was used successfully to measure DA in razor clams, mussels, scallops, and phytoplankton. The assay requires approximately 1.5 h to complete and has a standard 96-well format where each strip of eight wells is removable and can be stored at 4°C until needed. The first two wells of each strip serve as an internal control eliminating the need to run a standard curve. This allows as few as 3 or as many as 36 duplicate samples to be run at a time enabling real-time sample processing and limiting degradation of DA, which can occur during storage. There was minimal cross-reactivity in this assay with glutamine, glutamic acid, kainic acid, epi- or iso-DA. This accurate, rapid, cost-effective, assay offers environmental managers and public health officials an effective tool for monitoring DA concentrations in environment samples.

Time courses of intracellular and extracellular lipid classes in batch cultures of the toxic dinoflagellate, Gymnodinium cf. nagasakiense

Abstract The effect of temperature and light on lipid production was determined for an isolate of Gymnodinium cf. nagasakiense which is known to be toxic. Samples were taken in a time course in cultures grown at 13 or 18°C and at 35 or 75 μE·m −2 ·s −1 . The lipid class composition of Gymnodinium cf. nagasakiense cells and their exudates were analyzed with the Chromarod-Iatroscan system. The main intracellular lipid class, glycolipid, was present at similar concentrations in the cells in all three cultures. Maxima in intracellular concentrations of triacylglycerol, and minima in intracellular free fatty acid concentrations occurred near the time of maximum culture density. Dissolved free fatty acid concentrations were higher during the first 20 days than in the following 10 days of each culture. The culture grown at 13°C had the highest intracellular triacylglycerol concentration and the highest extracellular free fatty acid concentration. The culture grown at 35 μE·m −2 ·s −1 had the highest extracellular glycolipid concentration. Gas chromatographic analyses at the end of the growth experiments showed that pentaenoic fatty acids consisting mainly of 18:5n3 were twice as prominent in cells grown at the lower light level. However, the amount of 18:5n3 released into the surrounding medium in this culture was negligible.

Intra- and extracellular lipids in cultures of the toxic dinoflagellate, Gyrodinium aureolum

Abstract Gyrodinium aureolum is a North Atlantic red tide species of dinoflagellate which has frequently caused mortality of marine organisms in coastal waters and is a serious problem for the European fish farming industry. We describe here, for the first time, the lipid composition of extracts of cultures of an isolate of G. aureolum which is known to be toxic. Large volume samples were taken in the stationary phase of cultures grown at 13° and 18°. The lipids in extracts of G. aureolum cells and their exudates were analysed using TLC-FID, GC-FID and MS-MS. Polar lipids, including glycolipids and phospholipids, were the major type of lipids within the cells and in the exudates. Triacylglycerols were also prominent within the cells, while free fatty acids were prominent in the exudates. All- cis -3,6,9,12,1 5-octadecapentaenoic acid (18: 5n3) comprised 12–18% of the fatty acids in cell extracts and was the major unsaturated fatty acid present in cells from the warmer culture. The extracellular lipids contained considerably more monoeneoic, dieneoic and triencoic fatty acids than did the intracellular lipids, but had lower amounts of other unsaturated fatty acids.

Toxicity of fatty acid 18:5n3 from gymnodinium cf.mikimotoi: II. intracellular pH and K+ uptake in isolated trout hepatocytes

Effects of octadecapentaenoic acid 18:5n3 and other related polyunsaturated fatty acids present in Gymnodinium cf. mikimotoi were tested in isolated trout hepatocytes. These exotoxins decreased intracellular pH followed by a slow recovery to initial value and alkalinization of acidic compartments, suggesting an inhibition of vacuolar H+‐ATPases. Moreover, addition of 18:5n3 to the extracellular medium induced a decrease of K+ uptake into hepatocytes as a result of Na,K‐ATPase inhibition. However, high concentrations (10−5–10−3 M) are necessary to induce these effects. Copyright

Determination of glycoglycerolipids by Chromarod thin-layer chromatography with Iatroscan flame ionization detection

Abstract A Chromarod thin-layer chromatography separation procedure was developed for polar lipids in spinach, the flagellate Isochrysis galbana , and the toxic dinoflagellate, Gymnodinium sp. Monogalactosyl diacylglycerol, digalactosyl diacylglycerol and sulphoquinovosyl diacylglycerol were separated from each other, as well as from chlorophyll a , carotenoids, monoacylglycerol, phosphatidyl ethanolamine and other phospholipids. Quantitation of 0.5–10 μg loads of individual glycoglycerolipids and pigments was performed by scanning the rods through the flame ionization detector of an Iatroscan. Polar lipid class proportions in spinach and I. galbana were within 7% of those in the literature, and summed classes are 84% of total lipids by gravimetry.

Separation of polyunsaturated and saturated lipids from marine phytoplankton on silica gel-coated Chromarods

Abstract Observations of peak splitting in Chromarod separations of extracts of marine samples led to an in-depth study of this phenomenon. By co-spotting standards with lipids from the phytoplankton Gyrodinium aureolum and developing in hexane-based solvent systems it was determined that triacylglycerol and free fatty acid peaks were split due to the presence of high levels of polyunsaturated species. The content of formic acid in the solvent system controlled the separation of saturated and polyunsaturated free fatty acids from each other and from triacylglycerols. The amount of diethyl ether controlled the separation of saturated and polyunsaturated triacylglycerols from each other and from more polar components. It was possible to quantify individual components of split peaks provided loads were kept below 3 μg to maximize separations between species. Iatroscan-measured calibration curves revealed a slightly lower response for polyunsaturated species when developed in hexane-based solvent systems. The proportion of polyunsaturated species determined by Iatroscan compared well with the proportion of polyunsaturated fatty acids determined by gas chromatography.

A haemolytic test to assay toxins excreted by the marine dinoflagellate Gyrodinium cf. Aureolum

Abstract The haemolytic properties of phytoplankton toxins have been already demonstrated in many red tides. Although the detection of red blood cell (RBC) lysis is straightforward, it is not always easy to choose the most suitable type of RBC and incubation time. We have investigated these two points in order to obtain reproducible and quantifiable results, by applying the test to known haemolytic molecules. The dose-response curve is sigmoidal. The spiking technique allows a lowering of the detectable haemolytic threshold. Saponin and Kanagawas haemolysin were used. Without previous concentration, the detection limit was at 4000 HU 1 −1 . A chloroform-methanol extraction allowed the detection limit to be lowered to 0.1 HU 1 −1 . This method has been applied to the detection of a marine dinoflagellate whose exotoxin is haemolytic. It may be used for in situ detection of any haemolytic substance.