Genetic relationships in bipolar species of the protist ciliate, Euplotes

Protists thrive in polar oceans, where they represent a major driving force for globally important biogeochemical cycles and a key food-web component. Their biogeography is frequently associated to bipolar patterns of distribution. Although conceptually well supported by apparently unrestricted migration rates, the experimental certification of these patterns copes with the protist paucity of morphological characters with taxonomic value and difficulties in applying conventional species concepts. We studied three marine species of the ciliate Euplotes, E. euryhalinus, E. nobilii, and E. petzi, for their bipolar distribution by comparing the SSU-rRNA gene sequences and mating interactions of Antarctic, Patagonian, and Arctic strains. Each species was analogously found not to carry significantly varied SSU-rRNA gene sequences, implying a common occurrence of trans-equatorial genetic mixing. However, mating analyses revealed significant inter-species differences. Scarce Antarctic x Arctic strain mating compatibility distinguished E. petzi from E. euryhalinus and E. nobilii, in which mating pairs between Antarctic and Arctic strains were successfully induced. Yet, E. nobilii was the only one of the two species to show cross-fertilizing and fertile mating pairs. Taking the biological concept of species as discriminatory, it was thus concluded that only E. nobilii warrants the definition of genuine bipolar species

Analysis of 16S rRNA and mxaF genes revealing insights into Methylobacterium niche-specific plant association

The genus Methylobacterium comprises pink-pigmented facultative methylotrophic (PPFM) bacteria, known to be an important plant-associated bacterial group. Species of this group, described as plant-nodulating, have the dual capacity of producing cytokinin and enzymes, such as pectinase and cellulase, involved in systemic resistance induction and nitrogen fixation under specific plant environmental conditions. The aim hereby was to evaluate the phylogenetic distribution of Methylobacterium spp. isolates from different host plants. Thus, a comparative analysis between sequences from structural (16S rRNA) and functional mxaF (which codifies for a subunit of the enzyme methanol dehydrogenase) ubiquitous genes, was undertaken. Notably, some Methylobacterium spp. isolates are generalists through colonizing more than one host plant, whereas others are exclusively found in certain specific plant-species. Congruency between phylogeny and specific host inhabitance was higher in the mxaF gene than in the 16S rRNA, a possible indication of function-based selection in this niche. Therefore, in a first stage, plant colonization by Methylobacterium spp. could represent generalist behavior, possibly related to microbial competition and adaptation to a plant environment. Otherwise, niche-specific colonization is apparently impelled by the host plant

Pole-to-Pole Gene Flow in Protozoan Ciliates

Microorganisms represent the smallest but arguably most important component of the ocean life. They are essential to all nutrient cycles because they form the bottom of the marine food chain and outnumber all other marine species by orders of magnitude. Sampling of remote and inaccessible habitats and large-scale genomic analysis have shown how little we know about the microbial life in the oceans, and how our poor knowledge of the marine chemistry and biology is preventing us from foreseeing the detrimental effects that a too rapidly changing world has on the oceans’ ecosystems. In this context, polar microorganisms are attracting particular interest because of their role in global-scale biogeochemical cycles, in particular the carbon dioxide exchange with the atmosphere (Falkowski et al. 2008). Considering this interest, planktonic and benthic microbial communities from Arctic and Antarctic areas have become the focus of more systematic samplingand rigorous analyses for their taxonomic, prokaryotic (bacterial) and eukaryotic (protist), biodiversity. A relevant result of these analyses was the finding of microbial species that, like a diverse range of plant and animal species (Lindberg 1991; Crame 1993), warrant the definition ‘‘bipolar’’ (or ‘‘anti-tropical’’), i.e. species represented by high-latitude populations physically separated in distribution across the tropics (Darling et al. 2000; Montresor et al. 2003; Brandt et al. 2007; Pawlowski et al. 2007). This concept of species bipolarity has inherently raised the intriguing question whether co-specific Antarctic and Arctic populations evolved independently since the effective separation (approximately 10–15 million years ago) between the Arctic and Antarctic cold-water provinces, or whether a trans-tropical gene flow ensures that these polar populations maintain genetic continuity (Darling et al. 2000). Morphological studies alone are clearly insufficient to address this question, due to recurrent phenomena of parallel or convergent morphological evolution that take place under similar environmental forces. Therefore, more solid grounds supporting the concept of species bipolarity have been obtained in some species of foraminifera and dinozoans from analysis of genetic variation in sequences of the small subunit (SSU) rRNA nuclear gene (Darling et al. 2000; Montresor et al. 2003; Brandt et al. 2007; Pawlowski et al. 2007). Nevertheless, unless the calibration of a molecular clock is supported by abundance in fossil records, as is the case in foraminifera (Pawlowski et al. 1997), also this genetic approach is impaired by the fact that the same DNA regions may evolve at different rates among closely related organisms. A solution to overcoming this challenge is provided by the well-defined, monophyletic group of ciliates which are ideal organisms for the analysis of the breeding structure of natural microbial populations and, therefore, for obtaining data which satisfy the interbreeding criterion on which the biological (Darwinian) concept of species is founded. Ciliates govern their gene exchanges through a unique sexual phenomenon, known as conjugation (or mating), in which two functionally hermaphroditic individuals unite temporarily in pairs for a mutual exchange of a ‘‘migratory’’ (‘‘male’’) gametic nucleus destined to fuse into a synkaryon with a ‘‘resident’’ (‘‘female’’) gametic nucleus. In addition, ciliates more than any other group of polar microorganisms can readily provide living laboratory material in virtually unlimited amounts, since every aquatic polar habitat hosts a large variety of ciliate species that are relatively easy to collect and expand into permanent clonal cultures (i.e., descendants from single wild-type specimens). Here we review results that have been obtained from breeding analyses of Antarctic, Fuegian and Arctic populations of the ciliate Euplotes nobilii. These results have provided compelling evidence that these populations are genetically interconnected by gene flow and form a unique interbreeding biological species. The description of these results is preceded by an outline of our current knowledge of ciliate biodiversity in the polar regions, and by a rationalization of the genetic mechanism of the mating types that represents the major driving force of ciliate breeding strategies

Soil microbial diversity affects the plant-root colonization by arbuscular mycorrhizal fungi

Terrestrial plants establish symbiosis with arbuscular mycorrhizal fungi (AMF) to exchange water and nutrients. However, the extent to which soil biodiversity influences such association remains still unclear. Here, we manipulated the soil microbial diversity using a "dilution-to-extinction" approach in a controlled pot microcosm system and quantified the root length colonization of maize plants by the AMF Rhizophagus clarus. The experiment was performed by manipulating the soil microbiome within a native and foreign soil having distinct physicochemical properties. Overall, our data revealed significant positive correlations between the soil microbial diversity and AMF colonization. Most importantly, this finding opposes the diversity-invasibility hypothesis and highlights for a potential overall helper effect of the soil biodiversity on plant-AMF symbiosis

The secondary metabolite euplotin c induces apoptosis-like death in the marine ciliated protist euplotes vannus

The sesquiterpenoid euplotin C is a secondary metabolite produced by the ciliated protist Euplotes crassus and provides a mechanism for damping populations of potential competitors. Indeed, E. crassus is virtually resistant to its own product while different non-producer species representing an unbiased sample of the marine, interstitial, ciliate diversity are sensitive. For instance, euplotin C exerts a marked disruption of different homeostatic mechanisms in Euplotes vannus. We demonstrate by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay that euplotin C quickly decreases viability and mitochondrial function of E. vannus with a very high efficacy and at micromolar potency. In addition, euplotin C induces apoptosis in E. vannus as 4,6-diamino-2-phenylindole and Terminal Transferase dUTP Nick End Labeling staining show the rapid condensation and fragmentation of nuclear material in cells treated with euplotin C. These effects occur without detectable permeabilisation or rupture of cell membranes and with no major changes in the overall morphology, although some traits, such as vacuolisation and disorganised microtubules, can be observed by transmission electron microscopy. In particular, E. vannus show profound changes of the mitochondrial ultrastructure. Finally, we also show that caspase activity in E. vannus is increased by euplotin C. These data elucidate the pro-apoptotic role of euplotin C and suggest a mechanism for its impact on natural selection.L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com

Coding genes and molecular structures of the diffusible signalling proteins (pheromones) of the polar ciliate, Euplotes nobilii

In protozoan ciliates, diffusible signalling proteins (pheromones) regulate the vegetative growth and mating interactions. Here, the coding genes and the structures of the encoded pheromones were studied in genetically distinct wild-type strains representing interbreeding Antarctic and Arctic populations of the marine ciliate Euplotes nobilii. Determination of seven allelic pheromone-coding DNA sequences revealed that an unusual extension and high structural conservation of the 5′ non-coding region are peculiar traits of this gene family, implying that this region is directly involved in the mechanism of pheromone gene expression, possibly through phenomena of intron splicing and/or frame-shifting. For four pheromones, the three-dimensional structures were determined by nuclear magnetic resonance spectroscopy in solution. These structures show that the pheromones represent a protein family which adapts to its polar environment by combining a structurally stable core of a three-helix bundle with extended polypeptide segments that are devoid of regular secondary structures and concomitantly show enhanced structural flexibility

Cytotoxic effects and apoptotic signalling mechanisms of the sesquiterpenoid euplotin C, a secondary metabolite of the marine ciliate Euplotes crassus, in tumour cells

Most antitumour agents with cytotoxic properties induce apoptosis. The lipophilic compound euplotin C, isolated from the ciliate Euplotes crassus, is toxic to a number of different opportunistic or pathogenic microorganisms, although its mechanism of action is currently unknown. We report here that euplotin C is a powerful cytotoxic and pro-apoptotic agent in mouse AtT-20 and rat PC12 tumour-derived cell lines. In addition, we provide evidence that euplotin C treatment results in rapid activation of ryanodine receptors, depletion of Ca2+ stores in the endoplasmic reticulum (ER), the release of cytochrome c from the mitochondria, activation of caspase-12, and activation of caspase-3, leading to apoptosis. Intracellular Ca2+ overload is an early event which induces apoptosis and is parallelled by ER stress and the release of cytochrome c, whereas caspase-12 may be activated by euplotin C at a later stage in the apoptosis pathway. These events, either independently or concomitantly, lead to the activation of the caspase-3 and its downstream effectors, triggering the cell to undergo apoptosis. These results demonstrate that euplotin C may be considered for the design of cytotoxic and pro-apoptotic new drugs.L'articolo è disponibile sul sito dell'editore http://www.springerlink.com