Electrochemical detection of the toxic dinoflagellate Alexandrium ostenfeldii with a DNA-biosensor

The steady rise of observations of harmful or toxic algal blooms throughout the world in the past decades constitute a menace for coastal ecosystems and human interests. As a consequence, a number of programs have been launched to monitor the occurrence of harmful and toxic algae. However, the identification is currently done by microscopic examination, which requires a broad taxonomic knowledge, expensive equipment and is very time consuming. In order to facilitate the identification of toxic algae, an inexpensive and easy-to-handle DNA-biosensor has been adapted for the electrochemical detection of the toxic dinoflagellate Alexandrium ostenfeldii. The detection of the toxic algae is based on a sandwich hybridisation, which is carried out on a disposable sensor chip. A set of two probes for the species specific identification of A. ostenfeldii was developed. The specificity of the probes could be shown in dot-blot hybridisation and with the DNA-biosensor. The sensitivity of the DNA-biosensor was optimised with respect to hybridisation temperature and NaCl-concentration and a significant increase of the sensitivity of the DNA-biosensor could be obtained by a fragmentation of the rRNA prior to the hybridisation and by adding a helper oligonucleotide, which binds in close proximity to the probes to the hybridisation

Temperature and light requirements for growth of two diatom species (Bacillariophyceae) isolated from an Arctic macroalga

In the present study, two abundant epiphyticdiatom taxa were isolated from the assimilation hairs ofthe brown macroalga Chordaria flagelliformis collected inthe Arctic Kongsfjorden (Spitsbergen, Norway), establishedas unialgal cultures and their growth rates determinedunder controlled photon fluence rate andtemperature conditions. Using morphological (light andscanning electron microscopy) and SSU rRNA gene databoth isolates (ROS D99 and ROS D125) were identifiedas members of a FragilariaSynedropsis clade. Themolecular data of ROS D99 and ROS D125 were notidentical to any other published sequence. While ROSD99 has been identified as Fragilaria barbararum mainlydue to the SEM characteristics, ROS D125 could not bedefinitely identified although morphological data speakfor Fragilaria striatula. Both diatom species showedsimilar growth rates at all temperatures and photon fluencerates tested. They grew well between 0 and 15Cwithoptimum temperatures of 1214C, but did not survive20C. Therefore, compared to Antarctic diatoms bothtaxa from Kongsfjorden can be characterised as eurythermalorganisms. Increasing photon fluence rates between2 and 15 lmol m2 s1 were accompanied with analmost twofold increase in growth rates, but photon fluencerates >15 lmol m2 s1 did not further enhancegrowth pointing to low light requirements. From thesedata optimum, minimum and maximum photon fluencerates and temperatures for growth can be assessed indicatingthat both diatoms are well acclimated to the fluctuatingenvironmental conditions in the Arctic habitat

Bolidomonas: a new genus with two species belonging to a new algal class, the Bolidophyceae (Heterokonta) 1.

International audienceA new algal class, the Bolidophyceae (Heterokonta), is described from one genus, Bolidomonas, gen, nov., and two species, Bolidomonas pacifica, sp, nov and Bolidomonas mediterranea, sp, nov., isolated from the equatorial Pacific Ocean and the Mediterranean Sea, respectively. Both species are approximately 1.2 mu m in diameter and have two unequal flagella; the longer flagellum bears tubular hairs, whereas the shorter is smooth. The flagellar basal apparatus is restricted to two basal bodies, and there is no transitional helix. Cells are naked, devoid of walls or siliceous structures. The internal cellular organization is simple with a single plastid containing a ring genophore and a girdle lamella, one mitochondrion with tubular cristae, and one Golgi apparatus close to the basal bodies. The Mediterranean and the Pacific species differ in the insertion angle between their flagella and their pattern of swimming, these differences possibly being linked to each other. Analyses of the SSU rDNA gene place the two strains as a sister group to the diatoms, Moreover, pigment analyses confirm this position, as fucoxanthin is found as the major carotenoid in both lineages. These data strongly suggest that the ancestral heterokont that gave rise to the diatom lineage was probably a biflagellated unicell

Uncovering hidden biodiversity in the Cryptophyta: Clone library studies at the Helgoland Time Series Site in the Southern German Bight indentifies the cryptophycean clade potentially responsible for the majority of its genetic diversity during the spring bloom.

Cryptophyceae are important group in marine phytoplankton, but little is known about the occurrence and distribution of individual species. Recently, with use of molecular probes and microarray technology, it has been shown that species related to teleaulax spp. or Chroomonas spp. (clades 4 or 6) contributed the most to cryptophycean biomass in the north Sea. for the microarray study, the single probe (clade 4/6) recognizes members of both clades 4 and 6 and thus cannot separate them. Therefore, it was unknown as to whether the cryptophyte community was composed of clade 4, clade 6 or both of them. Here, we addressed this question and increased the genetic diversity of our investigations of cryptophycean diversity in the north Sea by sequencing 18S rRnA clone libraries made from fractionated water samples to examine specifically the picoplanktonic fraction because that fraction was studied in detail in the earlier microarray study. We focused on samples from the spring phytoplankton bloom in 2004 because the microarray signals were the strongest at this time. Excluding chimeric sequences, we detect- ed nine cryptophycean oTUs, seven of which fell into the teleaulax/Plagioselmis branch, whereas two grouped with Geminigera spp. our results indicate that these oTUs, affiliated with clade 4, may be an important component of cryptophyte community during spring bloom in the north Se

MIDTAL (Microarrays for the Detection of Toxic Algae)

Microalgae in marine and brackish waters of Europe regularly cause harmful effects, considered from the human perspective, in that they cause 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, present day monitoring is time consuming and based on morphology as determined by light microscopy, which may be insufficient to give definitive species and toxin attribution. In the European Union (EU) FP7 project MIDTAL (microarrays for the detection of toxic algae), we will first target rapid species identification using rRNA genes. The variable regions of the rRNA genes can be used for probe design to recognize species or even strains. Second, a toxin based microarray will be developed that includes 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 sequence. Existing rRNA probes and antibodies for toxic algal species/strains and their toxins will be adapted and optimized for microarray use. The purpose of MIDTAL is to support the common fisheries policy and to aid the national monitoring agencies by providing new rapid tools for the identification of toxic algae and their toxins so they can comply with EU directive 91/1491/CEE to monitor for toxic algae, and reduce the need for the mouse bioassay

Advances in the Detection of Toxic Algae Using Electrochemical Biosensors

Harmful algal blooms (HABs) are more frequent as climate changes and tropical toxic species move northward, especially along the Iberian Peninsula, a rich aquaculture area. Monitoring programs, detecting the presence of toxic algae before they bloom, are of paramount importance to protect ecosystems, aquaculture, human health and local economies. Rapid, reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention as an alternative to the legally required but impractical microscopic counting-based techniques. Our electrochemical detection system has improved, moving from conventional sandwich hybridization protocols using di�erent redox mediators and signal probes with di�erent labels to a novel strategy involving the recognition of RNA heteroduplexes by antibodies further labelled with bacterial antibody binding proteins conjugated with multiple enzyme molecules. Each change has increased sensitivity. A 150-fold signal increase has been produced with our newest protocol using magnetic microbeads (MBs) and amperometric detection at screen-printed carbon electrodes (SPCEs) to detect the target RNA of toxic species. We can detect as few as 10 cells