Christine Gescher

Feasibility of Assessing the Community Composition of Prasinophytes at the Helgoland Roads Sampling Site with a DNA Microarray

ABSTRACT The microalgal class Prasinophyceae (Chlorophyta) contains several picoeukaryotic species, which are known to be common in temperate and cold waters and have been observed to constitute major fractions of marine picoplankton. However, reliable detection and classification of prasinophytes are mainly hampered by their small size and few morphological markers. Consequently, very little is known about the abundance and ecology of the members of this class. In order to facilitate the assessment of the abundance of the Prasinophyceae, we have designed and evaluated an 18S rRNA gene-targeted oligonucleotide microarray consisting of 21 probes targeting different taxonomic levels of prasinophytes. The microarray contains both previously published probes from other hybridization methods and new probes, which were designed for novel prasinophyte groups. The evaluation of the probe set was done under stringent conditions with 18S PCR fragments from 20 unialgal reference cultures used as positive targets. This microarray has been applied to assess the community composition of prasinophytes at Helgoland, an island in the North Sea where time series data are collected and analyzed daily but only for the nano- and microplankton-size fractions. There is no identification of prasinophytes other than to record them numerically in the flagellate fraction. The samples were collected every 2 weeks between February 2004 and December 2006. The study here demonstrates the potential of DNA microarrays to be applied as a tool for quick general monitoring of this important picoplanktonic algal group.

CONTRIBUTION OF THE CLASS CRYPTOPHYCEAE TO PHYTOPLANKTON STRUCTURE IN THE GERMAN BIGHT1

The class Cryptophyceae (Division Cryptophyta) contains ecologically relevant species, which are widespread in aquatic environments. However, classification, identification, and enumeration of cryptophytes are challenged by a morphology that must be usually examined with EM to permit species identification. The quantitative importance of this group has been revealed by HPLC data. But ecological information assessing the occurrence or seasonality of cryptophytes in the marine environment is still scarce. Molecular techniques allow for a refined assessment of taxonomically challenging taxa, such as the cryptophytes. In our laboratory, a Phylochip was developed to facilitate and refine the assessment of cryptophyte microalgae. Here, we present the results of an environmental study that took advantage of the Phylochip. The study was designed to elucidate the seasonality and contribution of cryptophytes to phytoplankton structure in the German Bight. The occurrence of cryptophytes in total plankton versus the picoplankton fraction was assessed with the Phylochip between the years 2004 and 2006. Our data indicate that cryptophytes are an important and constant contributor to phytoplankton structure of the German Bight, especially in the picoplankton fraction.

Evaluation of locked nucleic acids for signal enhancement of oligonucleotide probes for microalgae immobilised on solid surfaces

Biosensors and microarrays are powerful tools for species detection and monitoring of microorganisms. A reliable identification of microorganisms with probe-based methods requires highly specific and sensitive probes. The introduction of locked nucleic acid (LNA) promises an enhancement of specificity and sensitivity of molecular probes. In this study, we compared specificity and sensitivity of conventional probes and LNA modified probes in two different solid phase hybridisation methods: sandwich hybridisation on biosensors and on DNA microarrays. In combination with DNA-microarrays, the LNA probes displayed an enhancement of sensitivity, but also gave more false-positive signals. With the biosensor, the LNA probes showed neither signal enhancement nor discrimination of a single mismatch. In all cases, conventional DNA probes showed equal or better results than LNA probes. In conclusion, LNA technology may have great potential in methods that use probes in suspension and in gene expressions studies, but under certain solid surface-hybridisation applications, they do not improve signal intensity.

phylochipanalyser — a program for analysing hierarchical probe sets

The recent introduction of phylochips that contain molecular probes facilitates environmental microbial identification in a single experiment without previous cultivation. A set of probes recognizing species at different taxonomic levels is denoted as a hierarchical set. Application of hierarchical probe sets on a DNA microarray allows the assessment of biodiversity with different resolutions. It significantly increases the robustness of the results retrieved from phylochip experiments because of the possible consistency checks of hybridization across different taxonomic levels. Here, we present a computer program, phylo‐chipanalyser, for the hierarchy editing and the evaluation of phylochip data generated from hierarchical probe sets.