New Approach Using the Real-Time PCR Method for Estimation of the Toxic Marine Dinoflagellate Ostreopsis cf. ovata in Marine Environment

Background: We describe the development and validation of a new quantitative real time PCR (qrt-PCR) method for the enumeration of the toxic benthic dinoflagellate Ostreopsis cf. ovata in marine environment. The benthic Ostreopsis sp. has a world-wide distribution and is associated during high biomass proliferation with the production of potent palytoxin-like compounds affecting human health and environment. Species-specific identification, which is relevant for the complex of different toxins production, by traditional methods of microscopy is difficult due to the high morphological variability, and thus different morphotypes can be easily misinterpreted. Methodology/Findings: The method is based on the SYBR I Green real-time PCR technology and combines the use of a plasmid standard curve with a ‘‘gold standard’’ created with pooled crude extracts from environmental samples collected during a bloom event of Ostreopsis cf. ovata in the Mediterranean Sea. Based on their similar PCR efficiencies (95% and 98%, respectively), the exact rDNA copy number per cell was obtained in cultured and environmental samples. Cell lysates were used as the templates to obtain total recovery of DNA. The analytical sensitivity of the PCR was set at two rDNA copy number and 8.061024 cell per reaction for plasmid and gold standards, respectively; the sensitivity of the assay was of cells g21 fw or 121 in macrophyte and seawater samples, respectively. The reproducibility was determined on the total linear quantification range of both curves confirming the accuracy of the technical set-up in the complete ranges of quantification over time. Conclusions/Significance: We developed a qrt-PCR assay specific, robust and high sample throughput for the absolute quantification of the toxic dinoflagellate Ostreopsis cf. ovata in the environmental samples. This molecular approach may be considered alternative to traditional microscopy and applied for the monitoring of benthic toxic microalgal species in the marine ecosystems

Phylogeography of Ostreopsis along West Pacific Coast, with Special Reference to a Novel Clade from Japan

BACKGROUND: A dinoflagellate genus Ostreopsis is known as a potential producer of Palytoxin derivatives. Palytoxin is the most potent non-proteinaceous compound reported so far. There has been a growing number of reports on palytoxin-like poisonings in southern areas of Japan; however, the distribution of Ostreopsis has not been investigated so far. Morphological plasticity of Ostreopsis makes reliable microscopic identification difficult so the employment of molecular tools was desirable. METHODS/PRINCIPAL FINDING: In total 223 clones were examined from samples mainly collected from southern areas of Japan. The D8-D10 region of the nuclear large subunit rDNA (D8-D10) was selected as a genetic marker and phylogenetic analyses were conducted. Although most of the clones were unable to be identified, there potentially 8 putative species established during this study. Among them, Ostreopsis sp. 1-5 did not belong to any known clade, and each of them formed its own clade. The dominant species was Ostreopsis sp. 1, which accounted for more than half of the clones and which was highly toxic and only distributed along the Japanese coast. Comparisons between the D8-D10 and the Internal Transcribed Spacer (ITS) region of the nuclear rDNA, which has widely been used for phylogenetic/phylogeographic studies in Ostreopsis, revealed that the D8-D10 was less variable than the ITS, making consistent and reliable phylogenetic reconstruction possible. CONCLUSIONS/SIGNIFICANCE: This study unveiled a surprisingly diverse and widespread distribution of Japanese Ostreopsis. Further study will be required to better understand the phylogeography of the genus. Our results posed the urgent need for the development of the early detection/warning systems for Ostreopsis, particularly for the widely distributed and strongly toxic Ostreopsis sp. 1. The D8-D10 marker will be suitable for these purposes

DNA barcoding and phylogenetic relationships in marine toxic dinoflagellate genus Ostreopsis based on mithocondrial genes

The marine dinoflagellate genus Ostreopsis includes species producing potent toxic compounds, such as palytoxin and palytoxin analogs, which cause toxic blooms in Mediterranean and tropical or other temperate areas. Phylogenetic and phylogeographical analyses based on ribosomal data set revealed the existence of distinct species and, within them, clades related to geographical distribution. Due to a high morphological variability, species belonging to this genus are very difficult to identify and cryptic species could be present. Thus, developing a standardized DNA barcode approach for this marine dinoflagellate can allow confirming known species and uncover hidden variability with consequent description of new species. These information, besides leading to a better understanding of species diversity in natural environment, could assist in identification and detection of different species directly in field sample. This study represents the first attempt to assess the suitability of mitochondrial COI (cytochrome c oxidase 1) and cob (cytochrome b) as an identification tool for Ostreopsis species. For this purpose, we designed specific primers to amplify and sequence mitochondrial COI and cob genes from several Ostreopsis spp. isolates from worldwide areas. Phylogenetic analyses of single and concatenated mtDNA genes within genus Ostreopsis, as well as their intra and inter-specific divergences, were compared to those based on nuclear ribosomal genes LSU and 5.8S-ITS regions. Phylogenetic analyses of the genus Ostreopsis using mtDNA genes resulted uninformative, being not able to distinguish different species. Moreover, the so called 'barcode gap' that is interspecific variation exceeding intraspecific variation, was detected only in ribosomal genes. Thus, our original goal to apply the mtDNA barcode to recognize species within this toxic marine dinoflagellate was not accomplishe

Complementary testing techniques applied to obtain the freeze-thaw resistance of concrete

Most of the standards that evaluate the resistance of concrete against freeze-thaw cycles (FTC) are based on the loss of weight due to scaling. Such procedures are useful but do not provide information about the microstructural deterioration of the concrete. The test procedure needs to be stopped after several FTCs for weighing the loss of material by scaling. This paper proposes the use of mercury-intrusion-porosimetry and thermogravimetric analysis for assessing the microstructural damage of concrete during FTCs. Continuous strain measurement can be performed without stopping the FTCs. The combination of the above techniques with the freeze-thaw resistance standards provides better and more precise information about concrete damage. The proposed procedure is applied to an ordinary concrete, a concrete with silica fume addition and one with an air-entraining agent. The test results showed that the three techniques used are suitable and useful to be employed as complementary to the standards.Peer reviewe