Picoplankton? We encounter them all the time. The tiniest drop of sea water is teeming with these organisms less than three microns in size.(1) Every time we take a dip, we are surrounded by billions of picoplanktonic cells.
These minute creatures – 25 years ago we did not even know they existed – remain an enigma. The European Picodiv project (Exploring the picoplankton diversity) aims to shed light on some of the mystery which surrounds this whole swathe of living creatures, concentrating in particular on phytosynthetic eucaryotes,(2) the picoplankton about which we know least of all.
Similar but different
'To date we have identified around 4 000 species of traditional phytoplankton. These are visible to the naked eye and commonly measure around 100 µm", as in the case of diatomea, for example, a very abundant species of unicellular algae,' explains Daniel Vaulot, a researcher at the Roscoff Biological Station in Roscoff, France and co-ordinator of the Picodiv project. 'But only about 30 species of picophytoplankton have been described. This is a derisory figure as we suspect there are a great many species of these truly minute organisms.' Picophagus flagellatus (described in 2000) measures around 2 µm, while the very abundant Prochlorococcus marinus, is among the smallest at just 0,5 µm.
To complicate matters, these oceanic lilliputians have the annoying tendency of resembling one another. Many are spherical in shape and green or brown in colour. This similarity does not, however, prevent them from displaying big differences in metabolism, environmental preferences, dynamism, pigments and other proteins. In short, they may look the same but they are in fact quite different.
'To describe a unicellular organism, in principle you have to be able to cultivate it,' continues Daniel Vaulot. 'The situation is further complicated with picoplankton because the usual nutritional environments always select the same species, which proliferate at the expense of those we are interested in. So we must take great pains to refine our culture mediums. We have achieved this with some success, but still have a long way to go.'
The latest molecular tools
Researchers are therefore using new techniques to try to penetrate the mysteries of picoplanktonic diversity, turning in particular to the latest advances in molecular biology. Carlos Pedros-Alio, of the Instituto de Ciencies del Mar in Barcelona, is one of the experts on these genetic tools with the Picodiv team. 'Even if a species is not described in the traditional sense, we can obtain a genetic signature,' he explains. 'We have succeeded in identifying very variable zones in the genome of these micro-organisms, more specifically in the sequence of the 18S ribosomal RNA gene. When two individuals show very different sequences in these zones, we can consider that they belong to different species, although it is not really very accurate to speak in terms of species for such minute creatures. When the differences exceed a certain limit, we consider they belong to distinct genres or classes.'
These signatures make it possible to detect changes in the composition of picoplanktonic communities. The so-called Denaturing Gradient Gel Electrophoresis (DGGE) technique is then applied. The 18S RNA gene is amplified in a seawater sample and deposited on a gel which is then subjected to an electric current causing the molecules to migrate. The result is a series of bands which form a fingerprint defining a given population. By using this tool, researchers are able to determine how, depending on the place and time of year, different picoplankton varieties succeed one another in the marine environment.
Also, it is now possible to count picoplankton, to define the density of a particular species. The method, known as Fish (Fluorescent In Situ Hybridisation) is quite simple. 'We prepare small quantities of DNA designed to fasten on to the signature of a given species,' continues Carlos Pedros-Alio. 'We then add a small fluorescent molecule. When this DNA solution is mixed with a sample taken from the ocean, a comparison between the proportion of fluorescent individuals and the total population (that can be counted under the microscope) gives us the proportion of the variety we are interested in.'
Bringing back the bounty
Finding out more about picoplankton could bring many benefits. There is certainly a fundamental aspect relating to knowledge of the life sciences. 'There are very complete catalogues of stars, some of which are millions of light years away,' stresses Carlos Pedros-Alio. 'It would be paradoxical not to know something about the populations of the water in which we bathe.'
Participants in the Picodiv project regularly work at three sampling sites, chosen as representative of the diversity of European coastal waters: Roscoff (Channel), Helgoland (North Sea) and the Bay of Blanes (Mediterranean). They also take part in a lot of marine expeditions, from the Arctic to the South Pacific. 'We sometimes have the impression we are working in the European tradition of the great 17th century naturalists who returned from every trip with new and extraordinary species and laid the foundations for the taxonomy of living organisms,' adds Carlos Pedros-Alio.
Important discoveries concerning the biology of this whole layer of life are already in prospect. According to Daniel Vaulot, ' heterotrophic eucaryotes (those without chlorophyll) used to be considered exclusively as predators. We now believe their role could be much more complex and varied, ranging from breaking down organic material to parasitism – none of which was suspected five years ago. In some waters, the picoplankton represent 80% of the biomass and play a determining ecological role at global level. Not only are they at the base of the food chain, but they are also involved in essential cycles of matter, especially of carbon or nitrogen. This means that a sound knowledge of the biology of picoplankton is essential for an accurate modelling of climate development.' When the associated species have been identified (in warmer waters or in waters rich in a given nutrient, for example), a simple sampling will tell us whether these indicator organisms are present.
Some species of picoplankton are also known to be toxic, so it is better to have tools with which to detect them.
'In this new field of research, co-operation between European laboratories gives us the critical mass needed to hold our own in the face of the US teams,' stresses Daniel Vaulot. 'In a sense, we are the pioneers of the study of small eucaryotes, a field that is proving very fruitful.' It is not for nothing that the work of the Picodiv researchers has been featured in the pages of Nature on a number of occasions.
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