Molecular detection of a potentially toxic diatom species.

Bidhan Dhar,Anna Brandi,Roberto Spurio,Stefania Marcheggiani,Kumar Singh,Letizia Brandi,Lucia Cimarelli,Camilla Puccinelli

doi:10.3390/ijerph120504921

Abstract

A few diatom species produce toxins that affect human and animal health. Among these, members of the Pseudo-nitzschia genus were the first diatoms unambiguously identified as producer of domoic acid, a neurotoxin affecting molluscan shell-fish, birds, marine mammals, and humans. Evidence exists indicating the involvement of another diatom genus, Amphora, as a potential producer of domoic acid. We present a strategy for the detection of the diatom species Amphora coffeaeformis based on the development of species-specific oligonucleotide probes and their application in microarray hybridization experiments. This approach is based on the use of two marker genes highly conserved in all diatoms, but endowed with sufficient genetic divergence to discriminate diatoms at the species level. A region of approximately 450 bp of these previously unexplored marker genes, coding for elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), was used to design oligonucleotide probes that were tested for specificity in combination with the corresponding fluorescently labeled DNA targets. The results presented in this work suggest a possible use of this DNA chip technology for the selective detection of A. coffeaeformis in environmental settings where the presence of this potential toxin producer may represent a threat to human and animal health. In addition, the same basic approach can be adapted to a wider range of diatoms for the simultaneous detection of microorganisms used as biomarkers of different water quality levels.

Highlights

Diatoms are single-celled photoautotrophic microorganisms present in virtually any habitat, though they are principally found in association with submerged surfaces or suspended in the water column of rivers, lakes, estuaries and oceans
The original sample consisted of a mixture of several benthic pennate diatom species, which were subjected to a multi-step procedure of dilution and washing until pure cultures derived from a single cell were obtained
In this study we describe the use of the sequence divergence of two marker genes, explored with the aim to by-pass the limitations of ribosomal RNA (rRNA) genes, for the development of species-specific oligonucleotides probes suitable for the detection of a diatom regarded as a potential biotoxin producer

Summary

Introduction

Diatoms are single-celled photoautotrophic microorganisms present in virtually any habitat, though they are principally found in association with submerged surfaces (benthic) or suspended in the water column of rivers, lakes, estuaries and oceans (planktonic). In addition to their contribution to net primary production, these algae can produce extracellular polymeric substances and storage polysaccharides (e.g., chrysolaminarin) and can accumulate a range of bioactive metabolites, such as PUFAs (poly-unsaturated fatty acids), which are an excellent food source for invertebrate grazers and harmful biotoxins [1,2,3]. The diatoms most frequently found associated with domoic acid production belong to the Pseudo-nitzschia genus, harmful algae isolated from many coastal and estuarine waters, among which those of United States and Canada [9], Portugal [10], Spain [11], France [12], Italy [13,14], Croatia [15], Greece [16], Ireland [17] and Australia [18,19]

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