In situ expression of eukaryotic ice-binding proteins in microbial communities of Arctic and Antarctic sea ice.

Ice-binding proteins (IBPs) have been isolated from various sea-ice organisms. Their characterisation points to a crucial role in protecting the organisms in sub-zero environments. However, their in situ abundance and diversity in natural sea-ice microbial communities is largely unknown. In this study, we analysed the expression and phylogenetic diversity of eukaryotic IBP transcripts from microbial communities of Arctic and Antarctic sea ice. IBP transcripts were found in abundances similar to those of proteins involved in core cellular processes such as photosynthesis. Eighty-nine percent of the IBP transcripts grouped with known IBP sequences from diatoms, haptophytes and crustaceans, but the majority represented novel sequences not previously characterized in cultured organisms. The observed high eukaryotic IBP expression in natural eukaryotic sea ice communities underlines the essential role of IBPs for survival of many microorganisms in communities living under the extreme conditions of polar sea ice.

Life-cycle modification in open oceans accounts for genome variability in a cosmopolitan phytoplankton.

Emiliania huxleyi is the most abundant calcifying plankton in modern oceans with substantial intraspecific genome variability and a biphasic life cycle involving sexual alternation between calcified 2N and flagellated 1N cells. We show that high genome content variability in Emiliania relates to erosion of 1N-specific genes and loss of the ability to form flagellated cells. Analysis of 185 E. huxleyi strains isolated from world oceans suggests that loss of flagella occurred independently in lineages inhabiting oligotrophic open oceans over short evolutionary timescales. This environmentally linked physiogenomic change suggests life cycling is not advantageous in very large/diluted populations experiencing low biotic pressure and low ecological variability. Gene loss did not appear to reflect pressure for genome streamlining in oligotrophic oceans as previously observed in picoplankton. Life-cycle modifications might be common in plankton and cause major functional variability to be hidden from traditional taxonomic or molecular markers.

An assessment of RNA content in Prymnesium parvum, Prymnesium polylepis, cf. Chattonella sp. and Karlodinium veneficum under varying environmental conditions for calibrating an RNA microarray for species detection.

Traditional methods of identification and enumeration can be somewhat ambiguous when identifying phytoplankton that requires electron microscopic examination to verify specific morphological features. Members of the genus Prymnesium (division Haptophyta), members of the Raphidophyceae and naked dinoflagellates are examples of such phytoplankton whose identification can be difficult. One alternative to traditional microscopy-based methods of identification is to use molecular protocols to detect target species. Methods that measure cellular DNA and RNA content can be used to estimate the number of cells present in a sample. This study investigated the variation of RNA yields in Prymnesium parvum, P. polylepis, cf. Chattonella sp. and Karlodinium veneficum cells grown under different light, temperature, salinity and inorganic nutrient conditions. This information was used to calibrate the signal intensity of a variety of oligonucleotide probes spotted onto the microarrays for the detection of toxic algae (MIDTAL), which is being developed to aid national monitoring agencies and to provide a faster means of identifying and quantifying harmful phytoplankton in water column samples.

Genome variations associated with viral susceptibility and calcification in Emiliania huxleyi.

Emiliania huxleyi, a key player in the global carbon cycle is one of the best studied coccolithophores with respect to biogeochemical cycles, climatology, and host-virus interactions. Strains of E. huxleyi show phenotypic plasticity regarding growth behaviour, light-response, calcification, acidification, and virus susceptibility. This phenomenon is likely a consequence of genomic differences, or transcriptomic responses, to environmental conditions or threats such as viral infections. We used an E. huxleyi genome microarray based on the sequenced strain CCMP1516 (reference strain) to perform comparative genomic hybridizations (CGH) of 16 E. huxleyi strains of different geographic origin. We investigated the genomic diversity and plasticity and focused on the identification of genes related to virus susceptibility and coccolith production (calcification). Among the tested 31940 gene models a core genome of 14628 genes was identified by hybridization among 16 E. huxleyi strains. 224 probes were characterized as specific for the reference strain CCMP1516. Compared to the sequenced E. huxleyi strain CCMP1516 variation in gene content of up to 30 percent among strains was observed. Comparison of core and non-core transcripts sets in terms of annotated functions reveals a broad, almost equal functional coverage over all KOG-categories of both transcript sets within the whole annotated genome. Within the variable (non-core) genome we identified genes associated with virus susceptibility and calcification. Genes associated with virus susceptibility include a Bax inhibitor-1 protein, three LRR receptor-like protein kinases, and mitogen-activated protein kinase. Our list of transcripts associated with coccolith production will stimulate further research, e.g. by genetic manipulation. In particular, the V-type proton ATPase 16 kDa proteolipid subunit is proposed to be a plausible target gene for further calcification studies.

Seasonal dynamics of harmful algae in outer Oslofjorden monitored by microarray, qPCR, and microscopy.

Monitoring of marine microalgae is important to predict and manage harmful algal blooms. Microarray Detection of Toxic ALgae (MIDTAL) is an FP7-funded EU project aiming to establish a multi-species microarray as a tool to aid monitoring agencies. We tested the suitability of different prototype versions of the MIDTAL microarray for the monthly monitoring of a sampling station in outer Oslofjorden during a 1-year period. Microarray data from two different versions of the MIDTAL chip were compared to results from cell counts (several species) and quantitative real-time PCR (qPCR; only Pseudochattonella spp.). While results from generation 2.5 microarrays exhibited a high number of false positive signals, generation 3.3 microarray data generally correlated with microscopy and qPCR data, with three important limitations: (1) Pseudo-nitzschia cells were not reliably detected, possibly because cells were not sufficiently retained during filtration or lysed during the extraction, and because of low sensitivity of the probes; (2) in the case of samples with high concentrations of non-target species, the sensitivity of the arrays was decreased; (3) one occurrence of Alexandrium pseudogonyaulax was not detected due to a 1-bp mismatch with the genus probe represented on the microarray. In spite of these shortcomings our data demonstrate the overall progress made and the potential of the MIDTAL array. The case of Pseudochattonella - where two morphologically similar species impossible to separate by light microscopy were distinguished - in particular, underlines the added value of molecular methods such as microarrays in routine phytoplankton monitoring.

Introduction to project MIDTAL: its methods and samples from Arcachon Bay, France.

Microalgae worldwide regularly cause harmful effects, considered from the human perspective, in that they cause health problems and economic damage to fisheries and tourism. Cyanobacteria cause similar problems in freshwaters. These episodes encompass a broad range of phenomena collectively referred to as "harmful algal blooms" (HABs). For adequate management of these phenomena, monitoring of microalgae is required. However, effective monitoring is time-consuming because cell morphology as determined by light microscopy may be insufficient to give definitive species and toxin attribution. In the European Union FP7 project MIDTAL (Microarrays for the Detection of Toxic Algae), we achieved rapid species identification using rRNA genes as the target. These regions can be targeted for probe design to recognise species or even strains. We also included antibody reactions to specific toxins produced by these microalgae because, even when cell numbers are low, toxins can be present and can accumulate in the shellfish. Microarrays are the state-of-the-art technology in molecular biology for the processing of bulk samples for detection of target RNA/DNA sequences. After 36 months, we have completed RNA-cell number-signal intensity calibration curves for 18 HAB species and the analysis of monthly field samples from five locations from year 1. Results from one location, Arcachon Bay (France), are reported here and compared favourably with cell counts in most cases. In general, the microarray was more sensitive than the cell counts, and this is likely a reflection in the difference in water volume analysed with the volume filtered for the microarray an order of magnitude greater.

Testing a Microarray to Detect and Monitor Toxic Microalgae in Arcachon Bay in France.

Harmful algal blooms (HABs) occur worldwide, causing health problems and economic damages to fisheries and tourism. Monitoring agencies are therefore essential, yet monitoring is based only on time-consuming light microscopy, a level at which a correct identification can be limited by insufficient morphological characters. The project MIDTAL (Microarray Detection of Toxic Algae)-an FP7-funded EU project-used rRNA genes (SSU and LSU) as a target on microarrays to identify toxic species. Furthermore, toxins were detected with a newly developed multiplex optical Surface Plasmon Resonance biosensor (Multi SPR) and compared with an enzyme-linked immunosorbent assay (ELISA). In this study, we demonstrate the latest generation of MIDTAL microarrays (version 3) and show the correlation between cell counts, detected toxin and microarray signals from field samples taken in Arcachon Bay in France in 2011. The MIDTAL microarray always detected more potentially toxic species than those detected by microscopic counts. The toxin detection was even more sensitive than both methods. Because of the universal nature of both toxin and species microarrays, they can be used to detect invasive species. Nevertheless, the MIDTAL microarray is not completely universal: first, because not all toxic species are on the chip, and second, because invasive species, such as Ostreopsis, already influence European coasts.