The pico is a very small unit, even smaller than the nano as it is the equivalent of 10-12. The biologist Aitor Alonso has devoted himself to studying green algae of this imperceptible size in the Bilbao estuary, paying particular attention to the area beyond the Nervión estuary. This has enabled him to identify six genera and eleven nano- and picoplanktonic species that until now had not been catalogued in these waters. He has also put forward some measures designed to optimize the methodology to be applied to these analyses.
His thesis, defended at the University of the Basque Country (UPV/EHU), is entitled Algas verdes en el picoplancton y nanoplancton del estuario del río Nervión (Golfo de Vizcaya) (Green algae in the picoplankton and nanoplankton in the estuary of the river Nervión (Bay of Biscay).
All the algae contain chlorophyll A, but it is chlorophyll B that characterises green algae and gives them their pigment. "They have been used for producing biofuels, for treating sewage, etc. Research has also been done on them in the quest for products for the pharmaceutical and cosmetics industries," says Alonso. But the practical applications of the green algae were not the starting point of this research, rather their extremely high relative presence among the smallest organisms in the Nervión estuary. "There is an abundance of them among the smallest algae in the plankton, in other words, picoplankton. Most of what can be found of that size, apart from bacteria, are green algae." So his main aim was to find out a little more about this specific diversity. Data like, for example, that the most abundant green algae belong to the chlorophyta division, which dominate in the outer part of this estuary during the summer.
Species and the TSA-FISH technique
The samples collected by Alonso between 2007 and 2010 have been used to isolate and analyse 26 algae strains from the estuary of the Nervión and other nearby ones. "We knew the green algae were a significant group, but not which species it actually consisted of," he explains. On the bases of 26 strains, he has been able to identity in his research six genera and eleven species: Eutreptiella (E. eupharyngeae and E. gymnastica), Mamiella (M. gilva), Nephroselmis (N. pyriformis), Pyramimonas (P. orientalis, P. moestrupii, P. grossii, P. robusta and P. propulsa), Oltmannsiellopsis (O. unicellularis and O. viridis) and cf. Chlamydomonas.
Alonso's main aim was to put a name to the species comprising the community of green algae in the estuary of Bilbao, but the path covered to achieve this has also been a significant contribution of this thesis. Studying organisms of such a tiny fraction like the picoplankton is complicated, and in this case the combination of three techniques was chosen: epifluorescence (an advanced alternative to the conventional optical microscope), flow cytometry (used to classify and count the cells that are found in fluids), and the TSA-FISH technique (which expands -- more than other techniques -- the fluorescent signals that work as algal markers, thus increasing sensitivity and facilitating analysis). The researcher has put forward a number of proposals to improve the performance of these techniques and to make a correct interpretation of the results obtained through them.
Alonso has concentrated, above all, on the problems of and possible solutions to the application of Tyramide Signal Amplification (TSA)-FISH. He concludes that in order to make the fitoplankton count as universal as possible, a protocol based on this technique needs to meet certain premises. For example, as the TSA-FISH has difficulties when it comes to doing the sample count, he recommends using flow cytometry for this task. The research also revealed shortcomings in the permeability of certain cultures analysed; that could lead to the importance of one or more microalgae groups, which could be in fact significant in the community, being overlooked. In order to avoid this problem, the proposal is to use an extra permeation through enzymatic treatment. With these measures TSA-FISH would allow a simple, quantitative, precise and reliable measurement to be made in addition to the monitoring of the phytoplanktonic populations.
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