Abstract
Our understanding of the composition of diatom communities and their response to environmental changes is currently limited by laborious taxonomic identification procedures. Advances in molecular technologies are expected to contribute more efficient, robust and sensitive tools for the detection of these ecologically relevant microorganisms. There is a need to explore and test phylogenetic markers as an alternative to the use of rRNA genes, whose limited sequence divergence does not allow the accurate discrimination of diatoms at the species level. In this work, nine diatom species belonging to eight genera, isolated from epylithic environmental samples collected in central Italy, were chosen to implement a panel of diatoms covering the full range of ecological status of freshwaters. The procedure described in this work relies on the PCR amplification of specific regions in two conserved diatom genes, elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), as a first step to narrow down the complexity of the targets, followed by microarray hybridization experiments. Oligonucleotide probes with the potential to discriminate closely related species were designed taking into account the genetic polymorphisms found in target genes. These probes were tested, refined and validated on a small-scale prototype DNA chip. Overall, we obtained 17 highly specific probes targeting eEF1-a and SIT, along with 19 probes having lower discriminatory power recognizing at the same time two or three species. This basic array was validated in a laboratory setting and is ready for tests with crude environmental samples eventually to be scaled-up to include a larger panel of diatoms. Its possible use for the simultaneous detection of diatoms selected from the classes of water quality identified by the European Water Framework Directive is discussed.
Highlights
Diatoms are microscopic unicellular algae characterized by the unique ability to engineer a mixture of silica, proteins and carbohydrates to form complex silica shells with well-defined architectural elements
These analyses, based on common criteria used by algologists to correlate physical and biological parameters with diatom assemblages [53,54], revealed a direct link between the presence and abundance of a selected panel of reliable diatom species and the water quality levels
This study was carried out within the collaborative “Universal Microarrays for the evaluation of fresh-water Quality based on detection of pathogens and their toxins” (MicroAQUA) project sponsored by the EU 7th Framework Programme
Summary
Diatoms are microscopic unicellular algae characterized by the unique ability to engineer a mixture of silica, proteins and carbohydrates to form complex silica shells with well-defined architectural elements. These photosynthetically active organisms contribute significantly to both terrestrial and marine primary productivity [1,2]. In addition to their enormous generation of organic carbon [3], diatoms play an ecologically relevant role in the global cycling of phosphorous, nitrogen and silica [4,5,6]. Several relevant studies have been conducted with the aim to correlate the quality of rivers and lakes through the use of indices based on diatoms [9,10,11,12,13]
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