Molecular tools for the selective detection of nine diatom species biomarkers of various water quality levels

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.

Keywords: diatoms; ecological status; freshwater; microarrays; oligonucleotide probes.

Figure 1

(A) Map of sampling sites in Regione Marche (Italy) and (B) list of diatoms species isolated and used in this work.

Figure 2

Photographs of the nine diatoms species used in this work: 1. Achnanthidium minutissimum; 2. Craticula halophila; 3. Cyclotella meneghiniana; 4. Eolimna minima; 5. Mayamaea atomus var. permitis; 6. Navicula veneta; 7. Nitzschia dissipata; 8. Nitzschia palea; 9. Surirella angusta. The pictures of diatom’s frustules were taken after removal of organic substances.

Figure 3

Electrophoretic separation of the amplification products obtained with primer pairs: (A) specific for SSU rRNA; (B) specific for a portion (440 bp) of the gene coding for the diatom elongation factor eEF1-a and (C) specific for a portion (470 bp) of the gene coding for the diatom silicic acid transporter gene (SIT). The panel of chromosomal DNA templates used corresponds to: 1. Nitzschia dissipata; 2. Nitzschia palea; 3. Navicula veneta; 4. Surirella angusta; 5. Craticula halophila; 6. Eolimna minima; 7. Achnanthidium minutissimum; 8. Mayamaea atomus var. permitis and 9. Cyclotella meneghiniana. Lane L contains the 100 bp DNA ladder Invitrogen (Panel A) and 100 bp DNA ladder Fermentas (Panel B and C).

Figure 4

Details of oligonucleotide probes designed on the amplicon corresponding to a portion of SIT gene of Craticula halophila. Black and grey arrows indicate sense probes and antisense probes, respectively.

Figure 5

Results of microarray hybridization experiments targeting SIT gene. (A) Image of an hybridized slide after scanning: the green dots indicate the Cy3-Marker spotted at the four corners of the slide, the green square indicates one of the four sectors of each block of the array. In the two graphs are reported the values of normalized signals obtained after hybridization with a fluorescent fragment of SIT gene from Craticula halophila (B) and Nitzschia palea (C). On the Y-axis are reported the probes producing a fluorescent signal after hybridization: the color code of the bars follows the scheme reported in Table S1 and in the text. Grey bars indicate false-positive signals.

Figure 6

Results of microarray hybridization experiments targeting eEF1-a gene. (A) Image of an hybridized slide after scanning: the green dots indicate the Cy3-Marker spotted at the four corners of the slide, the green square indicates one of the four sectors of each block of the array. In the two graphs are reported the values of normalized signals obtained after hybridization with a fluorescent fragment of eEF1-a gene from Cyclotella meneghiniana (B) and Navicula veneta (C). On the Y-axis are reported the probes producing a fluorescent signal after hybridization: the color code of the bars follows the scheme reported in Table S1 and in the text. Grey bars indicate false-positive signals.