DNA-Based Molecular Detection of Toxic Phytoplankton in Water Samples: Different Tools to Get Reliable Information

Abstract

The harmful phytoplankton holds sanitary, ecological and economic implications towards the human health, the coastal environments, and aquaculture facilities due to the consequences of recurrent harmful algal blooms (HABs). The adverse effects of HABs include toxin production, fish gill clogging, oxygen depletion and unpleasant water quality. The HABs are phenomena increasing worldwide for several reasons: eutrophication and/or unusual climatological conditions, the increased utilization of coastal waters for aquaculture, the movement of resting cysts caused by human activities (e.g. ships’ ballast waters or the translocation of shellfish stocks), and overfishing (1). Nowadays, it is essential to have rapid, sensitive and reliable methods to be applied in monitoring programs of marine coastal ecosystems for accurately and specifically detecting HAB species. Such methods can allow investigating the HAB species distribution and dispersion mechanism, and facilitating the prevention or mitigation of the harmful effects on human health, marine ecosystem and economic related activities. In routine monitoring programs, the detection and quantification of harmful species are based on morphological recognition through microscope analyses, which are time consuming and require considerable taxonomic expertise. In fact, the presence of morphologically similar species co-existing in the marine environments (e.g. Pseudo-nitzschia spp.) or different morphotypes of the same species can sometimes affect the monitoring reliability. Moreover, in some cases the fixation by Lugol or formalin can cause a morphological cellular distortion. Nevertheless, the fixation processes are crucial to preserve samples during the time intercurring between sample collection and laboratory analysis. Due to these limitations, many molecular methods for HAB species monitoring have been developed in the last years. Because of the instability of RNA (particularly mRNA) and proteins, most detection tools used for phytoplankton rely on detecting DNA. Target DNA sequences commonly used for developing specific primers and probes are rRNA genes, which are phylogenetically informative and tandemly repeated in high copy number. Molecular approaches to species identification and quantification may be broadly categorized as “whole cell” or “lysed cell” methods. In the “whole cell” methods the cells remain intact throughout sampling and processing (e.g. FISH); in the “lysed cell” methods, cells are disrupted and the resulting cell homogenate is analyzed, typically with subsequent calibration of the fluorescence or colorimetric signal back to cell number (e.g. sandwich hybridization, microarray hybridization, real-time qPCR). Advantages of the “lysed cell” approaches for bloom monitoring include amenability to automation and the possibility to analyze larger environmental sample volumes, thereby avoiding errors inherent in the small sample size used for whole cell methods. Disadvantages include the fact that it is not possible to visually inspect samples (e.g. to verify that the positive results are not due to crossreactions with non-target organisms), and that all sources of target sequences can contribute to the signal (including senescent/dead cells or cells obscured in food vacuoles or fecal pellets).