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Pseudo-nitzschia, or how a micro-alga becomes toxic

Some species of planktonic microscopic algae (phytoplankton) contain toxins which may cause serious health problems to humans, through the marine food chain. Among these is the diatom Pseudo-nitzschia which produces domoic acid, an amnesic shellfish toxin leading to potentially fatal food poisoning. The impact on consumers and the need to predict its blooms explain why it is necessary to better understand the factors playing a role on the production of toxins.

Pseudo-nitzschia strains were thus cultivated in the lab to study the relations between domoic acid production and physiological status of cells. Physiological status is described by several parameters, among which mortality rate, metabolic activity, lipid reserves, chlorophyll and photosynthesis efficiency.

Most of them are associated with a larger or smaller abundance of some molecules within cells. The principle of the method used is that, instead of measuring the amount of these molecules in a direct but complex and costly way, stains reacting specifically with them are added to the culture. These stains are fluorescent, i.e. when lighted they re-emit light in different, specific, wavelengths (colors). Measuring this re-emitted light intensity leads to estimate the concentration of molecules and therefore the physiological status of the cell. Fluorescence is measured through flow cytometry, a technique by which cells pass one by one in a laser beam at a very high rate. A major advantage of flow cytometry is to measure the physiological status of cells themselves and not of their whole population. These measurements were made daily during the 21-day development cycle of an algal culture, and were complemented by the monitoring of the bacterial population of the culture.

Lelong_fig3

Microscope observations of Pseudo-nitzschia cells. Each line shows a group of cells colored by a different fluorescent stains and observed in white light (left) and at two different wavelengths (center and right)

For the first time, technical protocols were defined to simultaneously measure a set of physiological parameters on micro-algal cells. Their application to Pseudo-nitzschia led to a better understanding of the physiological conditions of domoic acid production during a complete population growth cycle.

After a very slow start during the first week, Pseudo-nitzschia cultures grew exponentially, reaching a maximum after 17 to 19 days, after which the cell concentration rapidly declined. Cells stored lipid reserves during the initial lag phase, then used this source of energy for the needs of population growth; lipids then decreased and were once again accumulated when algal population peaked. Photosynthesis efficiency remained high during the whole exponential growth phase. Bacteria grew continuously in the cultures, but the bacteria/algae ratio was highest between 4 and 8 days.

Lelong_fig9

Principal component analysis is a way to summarize the information on all variables and observations. The closeness of the colored points (red: domoic acid concentration, green: stains fixed by lipids, blue: bacteria/algae ratio) shows the close relationships between these variables.

The evolution of all parameters through the development cycle of the culture was compared with that of domoic acid concentration, which was highest in the early exponential growth phase. This confirmed the hypotheses already put forward about the factors enhancing or decreasing Pseudo-nitzschia cell toxicity. Domoic acid is believed to be produced when cells have excess energy that is not used for primary metabolism; bacteria are also known to enhance its production through unknown mechanisms.

Beyond the application to a particular species of toxic alga, these results and the techniques used to obtain them will help to further the understanding of phytoplankton physiology and its responses to environmental changes, both biotic and abiotic.

 

The paper

Lelong A., Hégaret H., Soudant P., 2011. Cell-based measurements to assess physiological status of Pseudo-nitzschia multiseries, a toxic diatom. Research in Microbiology, 162 : 969-981.
See the first page

 

The authors

The three authors of this paper are members of the Laboratoire des sciences de l'environnement marin (Lemar) de l'IUEM.

 

The journal

In spite of its English title, Research in Microbiology is the direct descendant of the "Annales de l'Institut Pasteur", whose first issue in 1887 opened with a paper by Louis Pasteur entitled "Letter about rabies". Since then the journal changed a lot but never missed a monthly issue, even during the two World wars. Research in Microbiology publishes papers on fundamental and applied microbiology, covering all aspects of biology of bacteria, archaea and lower eukaryotes, and of their interactions (between them and with their environment). This paper was part of a special issue devoted to environmental microbiology.

 

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