Molecular analysis of the biodiversity of protists in hydrocarbon-polluted and pristine soils

Enrique Lara
EPFL, 2005
EPFL thesis

Eukaryotic microorganisms are an essential component of the Earth's global diversity. It is believed that the number of protistan taxa far outnumbers the one of viruses and bacteria. The taxonomic classification of these organisms, which also represent the major part of the total eukaryotic diversity, is still not very well known. It is only lately that the main phylogenetically consistent eukaryotic super assemblages (also called "kingdoms") have been defined. Inside these kingdoms, lateral branches represent the main multicellular taxa, the plants, animals and fungi. Protists are present in virtually all environments; in this thesis, attention was focused on the soil as habitat for the eukaryotic microorganisms. The communities inhabiting soil are characterized by adaptations to drought (the capacity to form cysts). Moreover, certain groups are found exclusively in this environment, to which they are morphologically adapted, whereas others are absent and exclusively found in aquatic systems. Detailed studies on the ecology of certain groups have been performed, and it is possible to define amongst them typical fast reproducing, early colonizers for new environments (r-specialists), whereas others can be found only in stable and highly productive habitats (K-specialists). All these organisms are distributed in a heterogeneous manner in the soil, being usually more frequent in the top horizon and around hotspots such as, for instance, root tips. Prokaryotes have drawn most of the attention of microbial ecologists, who almost neglected the eukaryotic microbes with the exception of fungi. Ecologists usually define four categories ("guilds") of protists in soil: Flagellates, naked amoebae, testate amoebae and ciliates. The importance of protistan activity in soil ecosystems has been shown with the concept of microbial loop: nutrients, produced by autotrophic organisms, are taken up by decomposers (bacteria and fungi) to produce their own biomass. Heterotrophic protists, by preying on them, release these compounds in a form which can be directly used by the autotrophs ("mineralization"), thus closing the loop. This phenomenon is of major significance, because protistan grazing controls the composition and activity of the prokaryotic community, and also the growth of the autotrophs. In soil, this phenomenon takes mainly place at the tip of the roots of vascular plants, where exudates are released. When soils are exposed to an organic pollution, the protistan communities and thus also their impact on the microbial loop, are influenced in two different ways: either by the direct toxic effects of the pollutant and or the indirect effects on their bacterial preys. A detailed study on the negative effects of environmental pollution, however, requires reliable methods to study protists. Molecular approaches offer the possibility to study protists in situ without the limitations inherent to cultivation dependent approaches. The aim of this thesis was to develop molecular methods and to apply these in combination with classical approaches for the characterization of protistan communities in a polycyclic aromatic hydrocarbon contamined and a non-contaminated control soil (PAH). The eukaryotic communities in a PAH polluted and a non-polluted control soil were first compared by amplifying total soil DNA with eukaryotic domain-specific primers targeting the 18S rRNA genes, cloning and sequencing the resulting PCR products. Using non-specific eukaryotic primers proved to be non-applicable for a reliable analysis of the 18S rRNA gene pools in soil, since protistan 18S rRNA gene sequences were (with the exception of the genus Acanthamoeba) underrepresented in the clone libraries obtained.. The biased picture obtained in the first chapter of the thesis was clearly shown when protist were cultivated from both soils: enrichment and cultivation of protists confirmed that members of the genus Acanthamoeba were indeed the most common protists in polluted soils, whereas they were completely absent from the control soil. Additionally, a diverse protistan community was recovered, which could not be detected by the molecular 18S rRNA-based approach. Twenty-two isolates of flagellates and naked amoebas were obtained, and characterised morphologically as well as by 18S rRNA gene sequence analysis. The diversity of cultivated organisms from the polluted soil was lower as compared to the control soil; however, the total number of protists was higher. Some of the cultures obtained in this cultivation dependant approach were used for further detailed studies; a jakobid isolate was described using light and electron microscopy, thus providing new insights into the knowledge of a relatively unknown group of excavates, i.e. the group of Jakoba incarcerata. Other cultures belonging to the kineoplastea were used to design a set of 18S rRNA-targeting fluorescently labelled oligonucleotide probes for fluorescence in situ hybridization. These probes are specific for the detection of the class Kinetoplastea and four genera within the class, i.e. Bodo, Parabodo, Rhynchomonas and Dimastigella and were applied to pure cultures and to environmental samples obtained by the so-called coverslip method. The effect of the PAH-pollution on a selected soil protistan guild, the ciliates, was further analyzed by applying ciliate-specific PCR based techniques. A specific primer set was used to amplify ciliate 18S rRNA genes from the environment and to conduct comparative sequence analysis of environmental clone libraries. This study indicated that the ciliate 18S rRNA gene pools present in the polluted soil were less diverse than those of the control soil. They contained mainly representatives of the class Colpodea, a clade of r-selected ciliates, typical for unstable environments. The control soil was characterized by a variety of trophic specialists, including carnivores, cyanobacteria and diatom feeders, in contrast to the polluted soil, which harboured mainly sequences affiliating to bacterivores and generalists. Based on these results, we could deduce that the trophic webs are less complex in a PAH polluted soil in comparison to a pristine control. This observation was further confirmed when we studied a larger number of samples using a fingerprinting technique, i.e. denaturing gradient gel electrophoresis (DGGE) with ciliate specific PCR primers. The profiles obtained showed again a dominance of the members of the class Colpodea in polluted soils.

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