Silvia Casabianca

Intraspecific variability in the response of bloom-forming marine microalgae to changed climate conditions

Phytoplankton populations can display high levels of genetic diversity that, when reflected by phenotypic variability, may stabilize a species response to environmental changes. We studied the effects of increased temperature and CO2 availability as predicted consequences of global change, on 16 genetically different isolates of the diatom Skeletonema marinoi from the Adriatic Sea and the Skagerrak (North Sea), and on eight strains of the PST (paralytic shellfish toxin)-producing dinoflagellate Alexandrium ostenfeldii from the Baltic Sea. Maximum growth rates were estimated in batch cultures of acclimated isolates grown for five to 10 generations in a factorial design at 20 and 24°C, and present day and next century applied atmospheric pCO2, respectively. In both species, individual strains were affected in different ways by increased temperature and pCO2. The strongest response variability, buffering overall effects, was detected among Adriatic S. marinoi strains. Skagerrak strains showed a more uniform response, particularly to increased temperature, with an overall positive effect on growth. Increased temperature also caused a general growth stimulation in A. ostenfeldii, despite notable variability in strain-specific response patterns. Our data revealed a significant relationship between strain-specific growth rates and the impact of pCO2 on growth—slow growing cultures were generally positively affected, while fast growing cultures showed no or negative responses to increased pCO2. Toxin composition of A. ostenfeldii was consistently altered by elevated temperature and increased CO2 supply in the tested strains, resulting in overall promotion of saxitoxin production by both treatments. Our findings suggest that phenotypic variability within populations plays an important role in the adaptation of phytoplankton to changing environments, potentially attenuating short-term effects and forming the basis for selection. In particular, A. ostenfeldii blooms may expand and increase in toxicity under increased water temperature and atmospheric pCO2 conditions, with potentially severe consequences for the coastal ecosystem.

First finding of Ostreopsis cf. ovata toxins in marine aerosols.

Since the late 1990s, a respiratory syndrome has been repetitively observed in humans concomitant with Ostreopsis spp. blooms (mainly O. cf. ovata) in the Mediterranean area. Previous studies have demonstrated that O. cf. ovata produces analogues of palytoxin (ovatoxins and a putative palytoxin), one of the most potent marine toxins. On the basis of the observed association between O. cf. ovata blooms, respiratory illness in people, and detection of palytoxin complex in algal samples, toxic aerosols, containing Ostreopsis cells and/or the toxins they produce, were postulated to be the cause of human illness. A small scale monitoring study of marine aerosol carried out along the Tuscan coasts (Italy) in 2009 and 2010 is reported. Aerosols were collected concomitantly with O. cf. ovata blooms, and they were analyzed by both PCR assays and LC-HRMS. The results, besides confirming the presence of O. cf. ovata cells, demonstrated for the first time the occurrence of ovatoxins in the aerosol at levels of 2.4 pg of ovatoxins per liter of air. Given the lack of toxicological data on palytoxins by inhalation exposure, our results are only a first step toward a more comprehensive understanding of the Ostreopsis-related respiratory syndrome.

Population genetic structure and connectivity of the harmful dinoflagellate Alexandrium minutum in the Mediterranean Sea

The toxin-producing microbial species Alexandrium minutum has a wide distribution in the Mediterranean Sea and causes high biomass blooms with consequences on the environment, human health and coastal-related economic activities. Comprehension of algal genetic differences and associated connectivity is fundamental to understand the geographical scale of adaptation and dispersal pathways of harmful microalgal species. In the present study, we combine A. minutum population genetic analyses based on microsatellites with indirect connectivity (Ci) estimations derived from a general circulation model of the Mediterranean sea. Our results show that four major clusters of genetically homogeneous groups can be identified, loosely corresponding to four regional seas: Adriatic, Ionian, Tyrrhenian and Catalan. Each of the four clusters included a small fraction of mixed and allochthonous genotypes from other Mediterranean areas, but the assignment to one of the four clusters was sufficiently robust as proved by the high ancestry coefficient values displayed by most of the individuals (>84%). The population structure of A. minutum on this scale can be explained by microalgal dispersion following the main regional circulation patterns over successive generations. We hypothesize that limited connectivity among the A. minutum populations results in low gene flow but not in the erosion of variability within the population, as indicated by the high gene diversity values. This study represents a first and new integrated approach, combining both genetic and numerical methods, to characterize and interpret the population structure of a toxic microalgal species. This approach of characterizing genetic population structure and connectivity at a regional scale holds promise for the control and management of the harmful algal bloom events in the Mediterranean Sea.

Development of an oligonucleotide microarray for the detection and monitoring of marine dinoflagellates.

Harmful Algal Blooms (HABs), mainly caused by dinoflagellates and diatoms, have great economic and sanitary implications. An important contribution for the comprehension of HAB phenomena and for the identification of risks related to toxic algal species is given by the monitoring programs. In the microscopy-based monitoring methods, harmful species are distinguished through their morphological characteristics. This can be time consuming and requires great taxonomic expertise due to the existence of morphologically close-related species. The high throughput, automation possibility and specificity of microarray-based detection assay, makes this technology very promising for qualitative detection of HAB species. In this study, an oligonucleotide microarray targeted to the ITS1-5.8S-ITS2 rDNA region of nine toxic dinoflagellate species/clades was designed and evaluated. Two probes (45-47 nucleotides in length) were designed for each species/clade to reduce the potential for false positives. The specificity and sensitivity of the probes were evaluated with ITS1-5.8S-ITS2 PCR amplicons obtained from 20 dinoflagellates cultured strains. Cross hybridization experiments confirmed the probe specificity; moreover, the assay showed a good sensitivity, allowing the detection of up to 2 ng of labeled PCR product. The applicability of the assay with field samples was demonstrated using net concentrated seawater samples, un-spiked or spiked with known amounts of cultured cells. Despite the general application of microarray technology for harmful algae detection is not new, a peculiar group of target species/clades has been included in this new-format assay. Moreover, novelties regarding mainly the probes and the target rDNA region have allowed sensitivity improvements in comparison to previously published studies.

Quantification of the toxic dinoflagellate ostreopsis spp. by qPCR Assay in marine aerosol

We report the development and validation of a qPCR based method for estimation of the toxic benthic dinoflagellate Ostreopsis cf. ovata in the complex matrix of marine aerosol at Sant Andreu de Llavaneres beach (northwestern Mediterranean Sea). Toxic events in humans after inhalation or cutaneous contact have been reported during O. cf. ovata blooms and were attributed to palytoxin (PLTX)-like compounds produced by this microalga. Similar PCR efficiencies of plasmid and cellular environmental standard curves (98 and 100%, respectively) allowed obtaining the rDNA copy number per cell. The analytical sensitivity was set at 2 × 10(0) rDNA copy number and 8 × 10(-4) cell per reaction. Based on spiking experiments, we evaluated the aerosol filter inhibitory activity and recovery rate of cells from filters, then normalized the abundance data of toxic O. cf. ovata. The abundance in marine aerosol during the bloom varied in the range of 1-102 cells per filter. Analytical determinations were also applied to detect palytoxin in field samples. No palytoxin was detected in the aerosol filters, and the estimation of PLTX like-compound concentrations in microepiphytic assemblages varied between 0.1 and 1.2 pg/cell.

BARRUFETA BRAVENSIS GEN. NOV. SP. NOV. (DINOPHYCEAE): A NEW BLOOM-FORMING SPECIES FROM THE NORTHWEST MEDITERRANEAN SEA(1).

The present study describes a new dinoflagellate genus, Barrufeta N. Sampedro et S. Fraga gen. nov., with one new species, B. bravensis Sampedro et S. Fraga sp. nov., isolated from the Costa Brava (NW Mediterranean Sea). The dinoflagellate was characterized at the genus and species levels by LM and EM; LSU and internal transcribed spacer (ITS) rDNA sequences; and HPLC analyses of the pigments, fatty acids, and possible presence of toxins of several cultured strains. The new Barrufeta species is oval shaped (22–35 μm long and 16–25 μm wide) and dorsoventrally flattened. It possesses numerous small chloroplasts that radiate from two large equatorially located pyrenoids and is a typical peridinin‐containing dinoflagellate. The nucleus is in the anterior part of the epicone. The apical groove has a characteristic “Smurf‐cap” shape that runs counterclockwise on the epicone and terminates on its right posterior part. B. bravensis is similar to the previously described species Gyrodinium resplendens Hulburt in its external morphology, but the original report of the latter lacked a description of the complete shape of the apical groove. It is therefore likely that some of the G. resplendens species reported in the literature are Barrufeta since they possess a Barrufeta‐type apical groove. Fatty acids of Barrufeta were more similar to those of Karenia brevis than those obtained from other unarmored analyzed species including three species of Gymnodinium and Akashiwo sanguinea.

Genetic diversity of the genus Ostreopsis Schmidt: Phylogeographical considerations and molecular methodology applications for field detection in the Mediterranean Sea

Abstract This study reports some recent phylogeographical considerations on the genus Ostreopsis distribution worldwide, with particular attention to the Mediterranean Sea, and new recent advances on the quali-quantitiative detection of Ostreopsis species along coastal areas of the Mediterranean Sea based on the PCR and quantitative real time PCR (qrt-PCR) assays. It was found that O. cf. ovata is widely dispersed throughout tropical and warm temperate coastal areas. In the Atlantic/Mediterranean region it represents a panmictic population that is highly divergent from Indo-Pacific populations. Furthermore, we demonstrated that the developed qrt-PCR assay is specific, robust and high sample throughput for the quantification of the toxic O. cf. ovata in the environmental samples. This molecular approach may be considered alternative to traditional methods of microscopy and applied for the survey of benthic toxic microalgal species in marine ecosystems.

Monitoring toxic Ostreopsis cf. ovata in recreational waters using a qPCR based assay.

Ostreopsis sp. is a toxic marine benthic dinoflagellate that causes high biomass blooms, posing a threat to human health, marine biota and aquaculture activities, and negatively impacting coastal seawater quality. Species-specific identification and enumeration is fundamental because it can allow the implementation of all the necessary preventive measures to properly manage Ostreopsis spp. bloom events in recreational waters and aquaculture farms. The aim of this study was to apply a rapid and sensitive qPCR method to quantify Ostreopsis cf. ovata abundance in environmental samples collected from Mediterranean coastal sites and to develop site-specific environmental standard curves. Similar PCR efficiencies of plasmid and environmental standard curves allowed us to estimate the LSU rDNA copy number per cell. Moreover, we assessed the effectiveness of mitochondrial COI and cob genes as alternative molecular markers to ribosomal genes in qPCR assays for Ostreopsis spp. quantification.

The sxt Gene and Paralytic Shellfish Poisoning Toxins as Markers for the Monitoring of Toxic Alexandrium Species Blooms

Paralytic shellfish poisoning (PSP) is a serious human illness caused by the ingestion of seafood contaminated with saxitoxin and its derivatives (STXs). These toxins are produced by some species of marine dinoflagellates within the genus Alexandrium. In the Mediterranean Sea, toxic Alexandrium spp. blooms, especially of A. minutum, are frequent and intense with negative impact to coastal ecosystem, aquaculture practices and other economic activities. We conducted a large scale study on the sxt gene and toxin distribution and content in toxic dinoflagellate A. minutum of the Mediterranean Sea using both quantitative PCR (qPCR) and HILIC-HRMS techniques. We developed a new qPCR assay for the estimation of the sxtA1 gene copy number in seawater samples during a bloom event in Syracuse Bay (Mediterranean Sea) with an analytical sensitivity of 2.0 × 10° sxtA1 gene copy number per reaction. The linear correlation between sxtA1 gene copy number and microalgal abundance and between the sxtA1 gene and STX content allowed us to rapidly determine the STX-producing cell concentrations of two Alexandrium species in environmental samples. In these samples, the amount of sxtA1 gene was in the range of 1.38 × 10(5) - 2.55 × 10(8) copies/L and the STX concentrations ranged from 41-201 nmol/L. This study described a potential PSP scenario in the Mediterranean Sea.

Genome complexity of harmful microalgae

During the past decade, next generation sequencing (NGS) technologies have provided new insights into the diversity, dynamics, and metabolic pathways of natural microbial communities. But, these new techniques face challenges related to the genome size and level of genome complexity of the species under investigation. Moreover, the coverage depth and the short-read length achieved by NGS based approaches also represent a major challenge for assembly. These factors could limit the use of these high-throughput sequencing methods for species lacking a reference genome and characterized by a high level of complexity. In the present work, the evolutionary history, mainly consisting of gene transfer events from bacteria and unicellular eukaryotes to microalgae, including harmful species, is discussed and reviewed as it relates to NGS application in microbial communities, with a particular focus on harmful algal bloom species and dinoflagellates. In the context of genetic population studies, genotyping-by-sequencing (GBS), an NGS based approach, could be used for the discovery and analysis of single nucleotide polymorphisms (SNPs). The NGS technologies are still relatively new and require further improvement. Specifically, there is a need to develop and standardize tools and approaches to handle large data sets, which have to be used for the majority of HAB species characterized by evolutionary highly dynamic genomes.