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

Variance component estimation uncertainty for unbalanced data: Application to a continent-wide vertical datum

Variance component estimation (VCE) is used to update the stochastic model in least-squares adjustments, but the uncertainty associated with the VCE-derived weights is rarely considered. Unbalanced data is where there is an unequal number of observations in each heterogeneous dataset comprising the variance component groups. As a case study using highly unbalanced data, we redefine a continent-wide vertical datum from a combined least-squares adjustment using iterative VCE and its uncertainties to update weights for each data set. These are: (1) a continent-wide levelling network, (2) a model of the ocean’s mean dynamic topography and mean sea level observations, and (3) GPS-derived ellipsoidal heights minus a gravimetric quasigeoid model. VCE uncertainty differs for each observation group in the highly unbalanced data, being dependent on the number of observations in each group. It also changes within each group after each VCE iteration, depending on the magnitude of change for each observation group’s variances. It is recommended that VCE uncertainty is computed for VCE updates to the weight matrix for unbalanced data so that the quality of the updates for each group can be properly assessed. This is particularly important if some groups contain relatively small numbers of observations. VCE uncertainty can also be used as a threshold for ceasing iterations, as it is shown—for this data set at least—that it is not necessary to continue time-consuming iterations to fully converge to unity

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

Estimating Dengue Transmission Intensity from Case-Notification Data from Multiple Countries

Despite being the most widely distributed mosquito-borne viral infection, estimates of dengue transmission intensity and associated burden remain ambiguous. With advances in the development of novel control measures, obtaining robust estimates of average dengue transmission intensity is key for assessing the burden of disease and the likely impact of interventions.We estimated the force of infection (λ) and corresponding basic reproduction numbers (R0) by fitting catalytic models to age-stratified incidence data identified from the literature. We compared estimates derived from incidence and seroprevalence data and assessed the level of under-reporting of dengue disease. In addition, we estimated the relative contribution of primary to quaternary infections to the observed burden of dengue disease incidence. The majority of R0 estimates ranged from one to five and the force of infection estimates from incidence data were consistent with those previously estimated from seroprevalence data. The baseline reporting rate (or the probability of detecting a secondary infection) was generally low (<25%) and varied within and between countries.As expected, estimates varied widely across and within countries, highlighting the spatio-temporally heterogeneous nature of dengue transmission. Although seroprevalence data provide the maximum information, the incidence models presented in this paper provide a method for estimating dengue transmission intensity from age-stratified incidence data, which will be an important consideration in areas where seroprevalence data are not available