Katja Metfies

Detection of Phytoplankton with Nucleic Acid Sensors

A potential hand-held biosensor system for the in-situ analysis of toxic algae was developed during the EU-project ALGADEC. Identification of toxic algae is based on molecular probes that specifically target its rRNA. 17 taxon specific probe sets were developed for harmful algae that occur in three different coastal areas in Europe. A sandwich-hybridization and two labelled probes are used to detect the rRNA. A capture probe, immobilised on the biosensor, binds to RNA-strands isolated from the target organism. A second digoxigen-labelled probe binds also to the RNA-strands. After incubation with an antibody-enzyme complex directed against digoxigenin, a substrate is added and a redox-reaction takes place. The resulting electrical current is measured and the amount of bound rRNA is proportional to the electrical current. The adaptation to the sensor and the probe specificity tests were done using laboratory strains with closely related species to avoid false positives and to guarantee that only desired strains are detected. The signals from the different probes are recorded by a microcontroller unit. If a PC is connected to the system, an easy to operate software visualizes process data, graphic results, and the measured values will be stored on the hard disc. The main steps of the analysis process are executed automatically in the measurement device. Only a manual filtering, including a lysis procedure has to be done before the automatic measurement. The portable ALGADEC device is also capable to operate as a stand-alone system with

Protist Communities in Moored Long-Term Sediment Traps (Fram Strait, Arctic)–Preservation with Mercury Chloride Allows for PCR-Based Molecular Genetic Analyses

Here we present a pilot study demonstrating, that preservation with mercury chloride allows the application of PCR-based molecular methods for the characterization of marine protist communities collected with moored long-term sediment traps. They can provide information on pelagic protist communities by collecting sinking plankton from the upper water column all year-round, even in remote polar oceans. Assessment of small protist species from the nano- and picoplankton fractions in sedimented material by microscopy is extremely challenging or almost impossible. Hence, comprehensive studies of variability in protist community composition in moored long-term sediment traps are scarce. Considering that marine nano- and picoeukaryotes are ecologically very important, new approaches are urgently needed to investigate protists in the smallest size-fractions of moored long-term sediment trap samples. We applied the quick and cost-effective Terminal Restriction Length Polymorphism (T-RFLP) on a set of selected samples that were collected between 2000 and 2010 in September at a depth of ~ 300 m in the area of the “LTER (Long-Term Ecological Research) site HAUSGARTEN“ in the eastern Fram Strait (Arctic). The results of these analyses suggest a change in the trapped protist community after 2002 in this area. A comparison of 18S sequences obtained via 454-pyrosequencing from samples collected in the water column and mercury chloride preserved sediment traps in 2009 and 2010 suggests, that sediment traps might reflect the pelagic eukaryotic microbial biodiversity qualitatively. Furthermore, we have indication that preservation with mercury chloride does not severely change the nucleotide composition of 18S rRNA genes in long-term sediment traps. Overall, we suggest that preservation with mercury chloride is a key to open the door for molecular genetic analyses of long-term sediment trap samples, and that PCR-based molecular methods have a strong potential to become an important tool for comprehensive taxonomic analyses of protist- and bacterial communities in moored long-term sediment traps.

Impact of sequence processing and taxonomic classification approaches on eukaryotic community structure from environmental samples with emphasis on diatoms

Next‐generation sequencing is a common method for analysing microbial community diversity and composition. Configuring an appropriate sequence processing strategy within the variety of tools and methods is a nontrivial task and can considerably influence the resulting community characteristics. We analysed the V4 region of 18S rRNA gene sequences of marine samples by 454‐pyrosequencing. Along this process, we generated several data sets with QIIME, mothur, and a custom‐made pipeline based on DNAStar and the phylogenetic tree‐based PhyloAssigner. For all processing strategies, default parameter settings and punctual variations were used. Our results revealed strong differences in total number of operational taxonomic units (OTUs), indicating that sequence preprocessing and clustering had a major impact on protist diversity estimates. However, diversity estimates of the abundant biosphere (abundance of ≥1%) were reproducible for all conducted processing pipeline versions. A qualitative comparison of diatom genera emphasized strong differences between the pipelines in which phylogenetic placement of sequences came closest to light microscopy‐based diatom identification. We conclude that diversity studies using different sequence processing strategies are comparable if the focus is on higher taxonomic levels, and if abundance thresholds are used to filter out OTUs of the rare biosphere.

Das Phytoplankton im Überblick

Das Phytoplankton besteht aus im Meer treibenden einzelligen Pflanzen, die nur mithilfe eines Mikroskops oder durch moderne molekularbiologische Techniken identifizierbar sind. Phytoplankton tritt zeitweilig massenhaft in Planktonbluten auf, die auch giftig sein konnen. Phytoplankton leistet einen wichtigen Beitrag zur globalen Sauerstoffproduktion und zur Reduktion von Kohlendioxid aus der Atmosphare und ist von wachsender Bedeutung im Klimawandel.