CYANO RT-Microarray: A Novel Tool to Detect Gene Expression in Cyanobacteria

EU μAQUA made early warning systems for freshwater pathogens and toxins. Barcodes for each cyanobacterial toxin gene published before 2011 were designed and used in a microarray format to capture messenger RNA to detect toxin gene expression at early stages of bloom development. A reverse transcriptase (RT) microarray was developed to detect toxin expression, which had low expression levels. Probes immobilized on the microarray slide captured the mRNA and were extended directly on the microarray. RT extension incorporated fluorescently labeled oligonucleotides to ensure a high signal detected by the microarray scanner. The CYANO RT microarray was laboratory tested with known toxic cyanobacteria and field-tested. Hybridizations without RT extensions were barely detectable in the cultured strains. However, with RT extensions, hybridized mRNA was easily detected. Field samples were equally successful and consistent with companion studies from the same sites using HPLC/(MS-MS) (High Performance Liquid Chromotography/ Mass Spec). In some cases, amplified expression produced a signal with no detection of that toxin using chemical means. The RT microarray may be more sensitive than HPLC/MS-MS. Further studies are needed to determine if the RT-microarray is detecting a very low expression of the toxin genes and, hence, more sensitive as an early warning system predicting the toxin potential

Mini Review: Diatom species as seen through a molecular window

It has been accepted that we know less than 10% of the identified diversity in the marine microbial world and the diatoms are no exception. Even the species that we think we can easily recognize are often cryptic species, and even less is known of their life histories and spatial and temporal trends in their abundance and distribution. With new molecular and analytical techniques, we can advance our knowledge of a species to understand its morphological range, biogeographies and reproductive isolation. Moreover, some of molecular techniques are very sensitive. Depending on the species-level question(s) being asked, the molecular tools appropriate to answer them differ greatly

Molecular Techniques for the Detection of Organisms in Aquatic Environments, with Emphasis on Harmful Algal Bloom Species.

Molecular techniques to detect organisms in aquatic ecosystems are being gradually considered as an attractive alternative to standard laboratory methods. They offer faster and more accurate means of detecting and monitoring species, with respect to their traditional homologues based on culture and microscopic counting. Molecular techniques are particularly attractive when multiple species need to be detected and/or are in very low abundance. This paper reviews molecular techniques based on whole cells, such as microscope-based enumeration and Fluorescence In-Situ Hybridization (FISH) and molecular cell-free formats, such as sandwich hybridization assay (SHA), biosensors, microarrays, quantitative polymerase chain reaction (qPCR) and real time PCR (RT-PCR). Those that combine one or several laboratory functions into a single integrated system (lab-on-a-chip) and techniques that generate a much higher throughput data, such as next-generation systems (NGS), were also reviewed. We also included some other approaches that enhance the performance of molecular techniques. For instance, nano-bioengineered probes and platforms, pre-concentration and magnetic separation systems, and solid-phase hybridization offer highly pre-concentration capabilities. Isothermal amplification and hybridization chain reaction (HCR) improve hybridization and amplification techniques. Finally, we presented a study case of field remote sensing of harmful algal blooms (HABs), the only example of real time monitoring, and close the discussion with future directions and concluding remarks

Sequence Analysis Confirms a New Algal Class

International audienc

Application of the µAqua microarray for pathogenic organisms across a marine/freshwater interface

Monitoring drinking water quality is an important public health issue and pathogenic organisms present a particularly serious health hazard in freshwater bodies. However, many pathogenic bacteria, including cyanobacteria, and pathogenic protozoa can be swept into coastal lagoons and into near-shore marine environments where they continue to grow and pose a health threat to marine mammals and invertebrates. In this study, wetested the suitability of a phylochip (microarray for species detection) developed for freshwater pathogenic organisms to be applied to samples taken across a marine/freshwater interface at monthly intervals for two years. Toxic cyanobacteria and pathogenic protozoa were more numerous in a coastal lagoon than at the freshwater or marine site, indicating that this microarray can be used to detect the presence of these pathogens across a marine/freshwater interface and thus the potential for toxicity to occur within the entire watershed

A timescale for diatom evolution based on four molecular markers: reassessment of ghost lineages and major steps defining diatom evolution.

ABSTRACT. – Phylogenies and molecular clocks of the diatoms have largely been inferred from SSU rDNA sequences. A new phylogeny of diatoms was estimated using four gene markers SSU and LSU rDNA rbcL and psbA (total 4352 bp) with 42 diatom species. The four gene trees analysed with a maximum likelihood (ML) and Baysian (BI) analysis recovered a monophyletic origin of the new diatom classes with high bootstrap support, which has been controversial with single gene markers using single outgroups and alignments that do not take secondary structure of the SSU gene into account. The divergence time of the classes were calculated from a ML tree in the MultliDiv Time program using a Bayesian estimation allowing for simultaneous constraints from the fossil record and varying rates of molecular evolution of different branches in the phylogenetic tree. These divergence times are generally in agreement with those proposed by other clocks using single genes with the exception that the pennates appear much earlier and suggest a longer Cretaceous fossil record that has yet to be sampled. Ghost lineages (i.e. the discrepancy between first appearance (FA) and molecular clock age of origin from an extant taxon) were revealed in the pennate lineage, whereas those ghost lineages in the centric lineages previously reported by others are reviewed and referred to earlier literature

Mini Review: Molecular Techniques for Identification and Characterization of Marine Biodiversity

Molecular methods to detect organisms in the natural environment can be divided into whole cell and cell free formats. Whole cell methods are generally limited by the number of fluorochromes that can be detected, whereas cell free formats offer more possibilities for multiple species detection and multiple methods of detection. This mini review addresses the major tools applied in environmental studies using cell free methods. The methods reviewed include microarrays, biosensors, quantitative real-time PCR (qPCR), and next generation sequencing (NGS)

Uncovering hidden biodiversity in the Cryptophyta: New picoplanktonic clades from clone library studies at the Helgoland time series site in the southern German Bight.

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 and 6) contributed most to cryptophyceam biomass in the North Sea. The probe for clades 4 and 6 cannot separate them and the single probe recognises members of both clades. Here, we increase 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 and to determine whether clade 4 or 6 were the dominant cyrptophytes. 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 detected 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 Sea

Obituary – Greta A. Fryxell

Noted oceanographer, female pioneer in academia, Professor Greta Albrecht Fryxell died from congestive heart failure on September 24, 2017 at her home in Claremont, Ca., USA at the age of 90

Review of the phylogenetic reconstruction of the diatoms using molecular tools with an analysis of a seven gene data set using multiple outgroups and morphological data for a total evidence approach

Medlin tested multiple outgroups with 18S rRNA dataset and found that haptophytes, ciliates, prasinophytes and chlorophytes recovered monophyletic Coscinodiscophyceae, Mediophyceae, Bacillariophyceae with strong BT support. Theriot et al. added six plastid genes to the diatom dataset but with only one outgroup, Bolidomonas and omitted most of the V4 region of that gene and bases beyond position 1200. They recovered a grade of clades from radial into polar centrics, into araphid pennates into the monophyletic raphid pennates. Their structural gradation hypothesis (SGH) contrasts to the CMB hypothesis of Medlin and Kaczmarska. We selected only those species with all seven genes from their dataset and added the entire 18S RNA gene to make a new dataset to which we sequentially added heterokont, haptophyte, and prasinophyte/chlorophyte outgroups. We analysed it using 1) evolutionary models with parameters relaxed across genes and codon positions for coding sequences (codon partition analysis scheme = CP) and 2) no partitions or evolutionary models as applied to each gene, using only optimised models of evolution for the entire dataset (NCP). CP recovered a monophyletic mediophycean and bacillariophycean clade and three coscinodiscophycean clades. Sequentially adding more outgroups did not change clade topology but dramatically increased BT support. NCP recovered a monophyletic Coscinodiscophyceae and Bacillariophyceae and three Mediophyceae clades, each with strong bootstrap support. Morphological data was added and analyzed similarly. NCP recovered three monophyletic classes and CP recovered the Bacillariophyceae arising from within the Mediophyceae, making the subphylum monophyletic but the class was paraphyletic. Each analysis was tested with SH tests in PAUP and IQTree. Plastid inheritance in the diatoms is not homogenous and thus their phylogenies may not be homologous. If so, then our application of gene models may be overparametrising the data. The application of no partitioning models with morphological data supported the CMB hypothesis