Manfred Jensen

Protistan community analysis: key findings of a large-scale molecular sampling

Protists are perhaps the most lineage-rich of microbial lifeforms, but remain largely unknown. High-throughput sequencing technologies provide opportunities to screen whole habitats in depth and enable detailed comparisons of different habitats to measure, compare and map protistan diversity. Such comparisons are often limited by low sample numbers within single studies and a lack of standardisation between studies. Here, we analysed 232 samples from 10 sampling campaigns using a standardised PCR protocol and bioinformatics pipeline. We show that protistan community patterns are highly consistent within habitat types and geographic regions, provided that sample processing is standardised. Community profiles are only weakly affected by fluctuations of the abundances of the most abundant taxa and, therefore, provide a sound basis for habitat comparison beyond random short-term fluctuations in the community composition. Further, we provide evidence that distribution patterns are not solely resulting from random processes. Distinct habitat types and distinct taxonomic groups are dominated by taxa with distinct distribution patterns that reflect their ecology with respect to dispersal and habitat colonisation. However, there is no systematic shift of the distribution pattern with taxon abundance.

Osmotic and atmospheric dehydration effects in the lichens Hypogymnia physodes, Lobaria pulmonaria, and Peltigera aphthosa: an in vivo study of the chlorophyll fluorescence induction.

Inactivation of photosynthesis during atmospheric and osmotic (highly concentrated NaCl or sucrose solutions) dehydration was monitored by measurement of chlorophyll fluorescence induction (OIP-phase, Kautsky-curves) in three lichen species. The induction curves were changed in a very similar way by all three treatments. All dehydration effects were rapidly reversible after rehydration. At relatively mild water stress, the rise time to the transient peak Fp was prolonged, and the variable part of fluorescence was diminished. In addition, at severe water stress, a considerable decline of the F0 value was observed. For NaCl treatment this effect started at water potentials <-8.5 MPa in P. aphthosa, <-12 MPa in H. physodes, and <-21 MPa in L. pulmonaria. Above these water potentials, our observations are in agreement with values from desiccation-tolerant algae, higher plants, and lichens, where an inactivation on the photosystem 2 (PS2) donor side has been postulated. At very low water potentials, the decrease in F0 probably monitors changes in the organization of the antenna apparatus of PS2.

CHLOROPHYLL FLUORESCENCE MEASUREMENTS IN THE FIELD: ASSESSMENT OF THE VITALITY OF LARGE NUMBERS OF LICHEN THALLI

Non-destructive measurement of photosynthetic activity as an indication of the health and potential growth of individual lichen thalli and of lichen populations.

Quantitative Variability of Direct Chemical Defense in Primary and Secondary Leaves of Lima Bean (Phaseolus lunatus) and Consequences for a Natural Herbivore

Ontogenetic variability in chemical plant defenses against herbivores is a common phenomenon, but the effects of this variability on herbivore–plant interactions are little understood. In a previous study on lima bean (Phaseolus lunatus), we found a trade-off between cyanogenesis, a direct defense, and the release of herbivore-induced volatile organic compounds (VOCs; mainly functioning as an indirect defense). Moreover, the expression of these two defenses could change during plant ontogeny. The present study aimed at elucidating whether such ontogenetic changes in plant defense can affect herbivore–plant interactions. We quantified feeding rates of a natural insect herbivore, the Mexican bean beetle (Epilachna varivestis), on primary and secondary leaves of individual lima bean plants. These insects strongly preferred low cyanogenic primary leaves over high cyanogenic secondary leaves. Although weakly defended by cyanogenesis, lima beans’ primary leaves showed protein concentrations and photosynthetic activities that did not differ significantly from secondary leaves at the time of analysis. Based on our findings, we suggest that lima beans’ long-lived primary leaves function as efficient source organs, even beyond the stage of seedlings. This hypothesis may explain why primary leaves express a strong indirect defense by the release of herbivore induced VOCs.

Measurement of Chlorophyll Fluorescence in Lichens

Chlorophyll fluorescence (CF) is the absorption of blue or red photons (= excitation) by chlorophyll molecules and the emission of far red photons (the energy difference is lost as heat). In contrast to chloroform solutions of chlorophyll or to fluorescent dyes, the emission from active photosynthetic systems does not remain constant, but changes in a complicated way after a constant excitation light is switched on. These changes (= variable fluorescence) are due to the onset of photosynthetic reactions that consume absorbed quanta. Accordingly, several derived fluorescence parameters (see below) provide diagnostic information about the state of the photosynthetic apparatus.

Trade‐off between taxon diversity and functional diversity in European lake ecosystems

Inferring ecosystem functioning and ecosystem services through inspections of the species inventory is a major aspect of ecological field studies. Ecosystem functions are often stable despite considerable species turnover. Using metatranscriptome analyses, we analyse a thus‐far unparalleled freshwater data set which comprises 21 mainland European freshwater lakes from the Sierra Nevada (Spain) to the Carpathian Mountains (Romania) and from northern Germany to the Apennines (Italy) and covers an altitudinal range from 38 m above sea level (a.s.l) to 3110 m a.s.l. The dominant taxa were Chlorophyta and streptophytic algae, Ciliophora, Bacillariophyta and Chrysophyta. Metatranscriptomics provided insights into differences in community composition and into functional diversity via the relative share of taxa to the overall read abundance of distinct functional genes on the ecosystem level. The dominant metabolic pathways in terms of the fraction of expressed sequences in the cDNA libraries were affiliated with primary metabolism, specifically oxidative phosphorylation, photosynthesis and the TCA cycle. Our analyses indicate that community composition is a good first proxy for the analysis of ecosystem functions. However, differential gene regulation modifies the relative importance of taxa in distinct pathways. Whereas taxon composition varies considerably between lakes, the relative importance of distinct metabolic pathways is much more stable, indicating that ecosystem functioning is buffered against shifts in community composition through a functional redundancy of taxa.

Cryptophyta as major bacterivores in freshwater summer plankton

Small bacterivorous eukaryotes play a cardinal role in aquatic food webs and their taxonomic classification is currently a hot topic in aquatic microbial ecology. Despite increasing interest in their diversity, core questions regarding predator–prey specificity remain largely unanswered, e.g., which heterotrophic nanoflagellates (HNFs) are the main bacterivores in freshwaters and which prokaryotes support the growth of small HNFs. To answer these questions, we fed natural communities of HNFs from Římov reservoir (Czech Republic) with five different bacterial strains of the ubiquitous betaproteobacterial genera Polynucleobacter and Limnohabitans. We combined amplicon sequencing and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) targeting eukaryotic 18 S rRNA genes to track specific responses of the natural HNF community to prey amendments. While amplicon sequencing provided valuable qualitative data and a basis for designing specific probes, the number of reads was insufficient to accurately quantify certain eukaryotic groups. We also applied a double-hybridization technique that allows simultaneous phylogenetic identification of both predator and prey. Our results show that community composition of HNFs is strongly dependent upon prey type. Surprisingly, Cryptophyta were the most abundant bacterivores, although this phylum has been so far assumed to be mainly autotrophic. Moreover, the growth of a small lineage of Cryptophyta (CRY1 clade) was strongly stimulated by one Limnohabitans strain in our experiment. Thus, our study is the first report that colorless Cryptophyta are major bacterivores in summer plankton samples and can play a key role in the carbon transfer from prokaryotes to higher trophic levels.

Quantitative Proteomics Reveals Ecophysiological Effects of Light and Silver Stress on the Mixotrophic Protist Poterioochromonas malhamensis

Aquatic environments are heavily impacted by human activities including climate warming and the introduction of xenobiotics. Due to the application of silver nanoparticles as bactericidal agent the introduction of silver into the environment strongly has increased during the past years. Silver ions affect the primary metabolism of algae, in particular photosynthesis. Mixotrophic algae are an interesting test case as they do not exclusively rely on photosynthesis which may attenuate the harmful effect of silver. In order to study the effect of silver ions on mixotrophs, cultures of the chrysophyte Poterioochromonas malhamensis were treated in a replicate design in light and darkness with silver nitrate at a sub-lethal concentration. At five time points samples were taken for the identification and quantitation of proteins by mass spectrometry. In our analysis, relative quantitative protein mass spectrometry has shown to be a useful tool for functional analyses in conjunction with transcriptome reference sequences. A total of 3,952 proteins in 63 samples were identified and quantified, mapping to 4,829 transcripts of the sequenced and assembled transcriptome. Among them, 720 and 104 proteins performing various cellular functions were differentially expressed after eight days in light versus darkness and after three days of silver treatment, respectively. Specifically pathways of the energy and primary carbon metabolism were differentially affected by light and the utilization of expensive reactions hints to an energy surplus of P. malhamensis under light conditions. The excess energy is not invested in growth, but in the synthesis of storage metabolites. The effects of silver were less explicit, observable especially in the dark treatments where the light effect could not mask coinciding but weaker effects of silver. Photosynthesis, particularly the light harvesting complexes, and several sulphur containing enzymes were affected presumably due to a direct interference with the silver ions, mainly affecting energy supply.

Evolution of heterotrophy in chrysophytes as reflected by comparative transcriptomics

ABSTRACT Shifts in the nutritional mode between phototrophy, mixotrophy and heterotrophy are a widespread phenomenon in the evolution of eukaryotic diversity. The transition between nutritional modes is particularly pronounced in chrysophytes and occurred independently several times through parallel evolution. Thus, chrysophytes provide a unique opportunity for studying the molecular basis of nutritional diversification and of the accompanying pathway reduction and degradation of plastid structures. In order to analyze the succession in switching the nutritional mode from mixotrophy to heterotrophy, we compared the transcriptome of the mixotrophic Poterioochromonas malhamensis with the transcriptomes of three obligate heterotrophic species of Ochromonadales. We used the transcriptome of P. malhamensis as a reference for plastid reduction in the heterotrophic taxa. The analyzed heterotrophic taxa were in different stages of plastid reduction. We investigated the reduction of several photosynthesis related pathways e.g. the xanthophyll cycle, the mevalonate pathway, the shikimate pathway and the tryptophan biosynthesis as well as the reduction of plastid structures and postulate a presumable succession of pathway reduction and degradation of accompanying structures. Figure. No Caption available.

Desiccation Stress and Fluorescence Imaging of the Peltigera Canina

Liquid water can restore the photosynthesis of most desiccated lichens within minutes. In contrast to lichens with green algal photobionts, cyanobacterial photobionts cannot uptake enough water from water vapor, i.e. they need liquid water to regain their photosynthetic activity (1). When exposed to wet air for several days, not even variable chlorophyll fluorescence can be detected in cyano-lichens (2). Obviously, their photosystem II reaction centres remain inactive. Moreover, the phycobilisomes remain functionally disconnected from photosystem II during this limited water uptake (3). In phycobilin (PB) fluorescence images (green excitation light), the fluorescence intensity becomes very low during desiccation (4), but until now there have been no data about the PB fluorescence intensity in the air-wetted state. Therefore we were very interested to know whether or not the PB fluorescence reappears in this phase. For this purpose we recorded PB fluorescence images in this and related states and compared the relative fluorescence intensity with chlorophyll fluorescence data obtained from an apparatus (MiniPAM) equipped with a small light guide (1.5 mm diameter). On the basis of 2-dimensional data, we discuss the surprising correlation between PB fluorescence intensity and the reappearance of variable chlorophyll fluorescence in some thallus regions. In addition, the role of the cortex + tomentum transparency and of the extent of pre-desiccation is assessed.