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Molecular probes for the detection and identification of ichthyotoxic marine microalgae of the genus Pseudochattonella (Dictyochophyceae, Ochrophyta)

  • Molecular tools for monitoring Harmful Algal Blooms
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Abstract

Phytoflagellates of the genus Pseudochattonella (Dictyochophyceae, Ochrophyta) form blooms in marine coastal waters in northern Europe, Japan, and New Zealand that at times cause fish kills with severe losses for the aquaculture industry. The aim of this study was to develop molecular probes for the detection and identification of Pseudochattonella at the genus and species level. A variety of probes were developed and applied to either dot blot hybridization, (q)PCR, or microarray format. In the dot blot hybridization assay, five different oligonucleotide probes targeting the small subunit (SSU) rDNA were tested against DNA from 18 microalgal strains and shown to be specific to the genus Pseudochattonella. A genus-specific PCR assay was developed by identifying an appropriate primer pair in the SSU—internal transcribed spacer 1 (ITS1) rDNA region. Its specificity was tested by screening against both target and non-target strains, and the assay was used to confirm the presence or absence of Pseudochattonella species in environmental samples. In order to distinguish between the two species of the genus, two PCR primer pairs each biased towards one of the species were designed in the large subunit (LSU) rDNA D1 domain and used for quantitative real-time PCR. Five selected probes (three SSU and two LSU rDNA) were adapted for the use on microarrays and included on a prototype multi-species microarray for the detection of harmful algae (http://www.midtal.com). Finally, microarrays and qPCR were used for the monthly monitoring of a sampling site in outer Oslofjorden during a 1-year period. Members of Pseudochattonella are difficult to identify by light microscopy in Lugol’s preserved samples, and the two species Pseudochattonella verruculosa and Pseudochattonella farcimen can be morphologically distinguished only by transmission electron microscopy. The molecular probes designed in this study will be a valuable asset to microscopical detection methods in the monitoring of harmful algae and for biogeographical and ecological studies of this genus.

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Acknowledgments

The development of microarray protocols as well as microarray specificity tests were coordinated by Linda K. Medlin and carried out and aided by partners of the MIDTAL project (http://www.midtal.com/partners.php). We also thank Jeanette Göbel for providing an algal culture of the strain P. verruculosa JG8 from the North Sea 2000, Daniel Vaulot for non-target strains from the Roscoff culture collection, Fumie Kasai for strain NIES 670 from the NIES culture collection, Jolanda M. van Iperen for providing an environmental sample from a bloom in the Netherlands in 2006, Uwe John for help with designing and testing an early version of the qPCR assay, Anette Engesmo for help with the presented qPCR experiments, and the reviewers for their helpful comments. IR was financially supported by University of Oslo strategic funding. MIDTAL is a project under the EU’s 7th Framework Program (FP7-ENV-2007-1-MIDTAL-201724) and provided funding for SMD and BE during this work.

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  1. Simon M. Dittami

    Present address: Station Biologique de Roscoff, UMR1739, Place Georges Teissier, 29680, Roscoff, France

Authors and Affiliations

  1. Department of Biology, Marine Biology, University of Oslo, P.O. Box 1066, Blindern, 0316, Oslo, Norway

    Simon M. Dittami, Ingvild Riisberg & Bente Edvardsen

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  1. Simon M. Dittami
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  2. Ingvild Riisberg
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  3. Bente Edvardsen
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Correspondence to Bente Edvardsen.

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Responsible editor: Robert Duran

Simon M. Dittami and Ingvild Riisberg contributed equally to this study.

Electronic supplementary material

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Supplementary file 1

Dot blot hybridizations of membrane-bound SSU rDNA with six DIG-labeled oligonucleotide probes as in Table 2. Amplified SSU rDNA (approximately 20–100 ng) from the following isolates was spotted on the membrane in the order: (1A–1E) P. farcimen UIO109-UIO113; (1F) P. verruculosa NIES 670 2A, P. verruculosa JG8; (2B) Florenciella parvula; (2C) Dictyocha speculum; (2D) Dictyochophyceae strain RCC 505; (2E) Dictyochophyceae strain RCC 381; (2F) Heterosigma akashiwo; (3A) Olisthodiscus luteus; (3B) Skeletonema sp.; (3C) Imantonia rotunda; (3D) Emiliania huxleyi; (3E) Dunaliella tertiolecta (PDF 1345 kb)

Supplementary file 2

Calibration curves for the two species-biased primer pairs targeting Pseudochattonella LSU rDNA as well as the general eukaryote primer pair (1400F and 1528R) targeting SSU rDNA used in the qPCR experiments (PDF 134 kb)

Supplementary file 3

Sample melting curves obtained for the P. farcimen and P. verruculosa LSU rDNA primers with a range of different strains (Table 1) and spiked environmental samples. All curves show a distinct peak at approximately 85.5–86 °C; no difference in melting temperature was detected between the two species (PDF 102 kb)

Supplementary file 4

Total quantity of DNA extracted from spiked field samples estimated from Nanodrop measurements (PDF 255 kb)

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Dittami, S.M., Riisberg, I. & Edvardsen, B. Molecular probes for the detection and identification of ichthyotoxic marine microalgae of the genus Pseudochattonella (Dictyochophyceae, Ochrophyta). Environ Sci Pollut Res 20, 6824–6837 (2013). https://doi.org/10.1007/s11356-012-1402-2

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