Antonella Penna

Development of a Real-Time PCR Assay for Rapid Detection and Quantification of Alexandrium minutum (a Dinoflagellate)

ABSTRACT The marine dinoflagellate genus Alexandrium includes a number of species which produce neurotoxins responsible for paralytic shellfish poisoning (PSP), which in humans may cause muscular paralysis, neurological symptoms, and, in extreme cases, death. A. minutum is the most widespread toxic PSP species in the western Mediterranean basin. The monitoring of coastal waters for the presence of harmful algae also normally involves microscopic examinations of phytoplankton populations. These procedures are time consuming and require a great deal of taxonomic experience, thus limiting the number of specimens that can be analyzed. Because of the genetic diversity of different genera and species, molecular tools may also help to detect the presence of target microorganisms in marine field samples. In this study, we developed a real-time PCR-based assay for rapid detection of all toxic species of the Alexandrium genus in both fixative-preserved environmental samples and cultures. Moreover, we developed a real-time quantitative PCR assay for the quantification of A. minutum cells in seawater samples. Alexandrium genus-specific primers were designed on the 5.8S rDNA region. Primer specificity was confirmed by using BLAST and by amplification of a representative sample of the DNA of other dinoflagellates and diatoms. Using a standard curve constructed with a plasmid containing the ITS1-5.8S-ITS2 A. minutum sequence and cultured A. minutum cells, we determined the absolute number of 5.8S rDNA copies per cell. Consequently, after quantification of 5.8S rDNA copies in samples containing A. minutum cells, we were also able to estimate the number of cells. Several fixed A. minutum bloom sea samples from Arenys Harbor (Catalan Coast, Spain) were analyzed using this method, and quantification results were compared with standard microscopy counting methods. The two methods gave comparable results, confirming that real-time PCR could be a valid, fast alternative procedure for the detection and quantification of target phytoplankton species during coastal water monitoring.

CHARACTERIZATION OF OSTREOPSIS AND COOLIA (DINOPHYCEAE) ISOLATES IN THE WESTERN MEDITERRANEAN SEA BASED ON MORPHOLOGY, TOXICITY AND INTERNAL TRANSCRIBED SPACER 5.8S rDNA SEQUENCES†

Several isolates of epiphytic dinoflagellates belonging to the genera Ostreopsis Schmidt and Coolia Meunier from the western Mediterranean Sea were examined by LM and EM, toxicity assays, and internal transcribed spacer (ITS) regions of nuclear rDNA, and 5.8S rDNA were sequenced. Morphological comparisons based on the analyses of cell shape, size, thecal plates, and surface ornamentation revealed two distinct species in the western Mediterranean: O. cf. siamensis Schmidt from the Catalan, Andalusian, and Sicilian coasts and O. ovata Fukuyo from the Ligurian coast, southern Tyrrhenian Sea, and Balearic Islands. Both Ostreopsis species were toxic; however, no differences in toxicity were detected between the two Ostreopsis species. Coolia monotis Meunier was nontoxic. The morphological studies were supported by phylogenetic analyses; all western Mediterranean isolates of O. cf. siamensis showed ITS and 5.8S rDNA sequences identical to each other and so did those of O. ovata, whereas high genetic diversity was detected between the western Mediterranean and Asian isolates of O. ovata. The nucleotide sequence analyses of the C. monotis strains showed that all C. monotis isolates from Europe formed a homogeneous clade. Further, the genetic diversity was high between the European and Asian C. monotis isolates. In this study, genetic markers combined with morphology and toxicity analyses was useful in the taxonomic and phylogenetic studies of the Ostreopsidaceae in a temperate area.

Unique Toxin Profile of a Mediterranean Ostreopsis cf. ovata Strain: HR LC-MSn Characterization of Ovatoxin-f, a New Palytoxin Congener

Currently, the benthic dinoflagellate Ostreopsis cf. ovata represents a serious concern to human health in the whole Mediterranean basin due to the production of palytoxin congeners, a putative palytoxin and ovatoxins (ovatoxin-a, -b, -c, -d/-e), listed among the most potent marine toxins. High resolution liquid chromatography-mass spectrometry (HR LC-MS) based investigation of a North Western Adriatic strain of Ostreopsis cf. ovata collected at Portonovo (Italy) in 2008 is reported herein. Toxin profile was different from those previously reported for other O. cf. ovata, both qualitatively and quantitatively. For the first time, ovatoxin-a did not dominate the toxin profile, and a new palytoxin congener, here named ovatoxin-f, was detected. Ovatoxin-f and its elemental formula present C(2)H(4) more than ovatoxin-a. HR CID MS(n) experiments allowed us to restrict structural differences between ovatoxin-a and -f to the region between C-95 and C-102, a region not previously been described to be modified in other palytoxins. Ovatoxin-f represents the major component of the toxin profile of the analyzed strain accounting for 50% of the total toxin content, while ovatoxin-a, the dominant toxin in most of the Mediterranean O. cf. ovata strains we have analyzed so far, is the second major component of the toxin profile (23%). Thus, the presence of ovatoxin-f should be taken into account when monitoring programs for palytoxin-like compounds in microalgae and/or seawater are carried out.

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.

IDENTIFICATION OF ALEXANDRIUM (DINOPHYCEAE) SPECIES USING PCR AND rDNA‐TARGETED PROBES

A PCR (polymerase chain reaction)‐based assay for the detection of Alexandrium species in cultured samples using rDNA‐targeted probes was developed. The internal transcribed spacers 1 and 2 (ITS1 and ITS2) and the 5.8S ribosomal RNA gene (rDNA) from cultured isolates of A. tamarense (Lebour) Taylor, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech and A. lusitanicum Balech were amplified using PCR and sequenced. Sequence comparisons showed that the 5.8S and ITS1‐ITS2 regions contain sequences specific for the Alexandrium genus, especially at the 3′ end of the 5.8S coding region. PCR primers and a radioactive 32P‐labeled DNA probe were devised for this region. The cross‐reactivity of the PCR primers and probe was tested against cultured isolates of Alexandrium and other dinoflagellates and diatoms. All the Alexandrium isolates screened reacted toward the genus‐specific probe; in contrast, the other groups of microalgae (dinoflagellates and diatoms) did not react with the probe. Furthermore, the PCR amplification technique combined with the use of the rDNA‐target probe allowed us to develop a method for the detection of Alexandrium cells in cultured samples. This PCR method might offer a new approach for the identification and enumeration of the HAB (harmful algal bloom) species present in natural phytoplankton populations.

Analysis of rRNA gene content in the Mediterranean dinoflagellate Alexandrium catenella and Alexandrium taylori: implications for the quantitative real-time PCR-based monitoring methods

A number of species belonging to the genus Alexandrium are among the main toxic microalgae responsible for Harmful Algal Blooms (HABs). The monitoring of coastal waters for the presence of these microalgae is essential to identify correlations between cell abundances and environmental factors that regulate bloom dynamics. In the attempt to improve the monitoring sensitivity and the rapidity at which a large number of field samples can be processed, several molecular methods for the detection of genetically distinct HAB species have been developed during the last years. In particular, real-time PCR has been shown to be a powerful method for quantitative detection of HAB species in environmental samples. When a plasmid is used as a standard, the knowledge of the amount of target gene per cell is essential for the determination of the cell number in the field sample. In this study, we analyzed the rRNA gene content variability in several Alexandrium catenella and Alexandrium taylori strains isolated from the Mediterranean Sea using a real-time PCR-based approach. The rRNA gene content was also analyzed in different growth phases, from early exponential to stationary conditions. The results showed a general variability in the rRNA gene content depending on the strain and, for the species A. taylori, in relation also to the growth phase. These results should be taken into account for the application of the real-time quantitative PCR-based techniques for monitoring purposes in coastal seawaters.

Ostreopsis cf. ovata bloom in the northern Adriatic Sea during summer 2009: Ecology, molecular characterization and toxin profile

Intense blooms of the benthic dinoflagellate Ostreopsis cf. ovata have occurred in the northern Adriatic Sea since 2006. These blooms are associated with noxious effects on human health and with the mortality of benthic organisms because of the production of palytoxin-like compounds. The O. cf. ovata bloom and its relationships with nutrient concentrations at two stations on the Conero Riviera (northern Adriatic Sea) were investigated in the summer of 2009. O. cf. ovata developed from August to November, with the highest abundances in September (1.3×10(6) cells g(-1) fw corresponding to 63.8×10(3) cells cm(-2)). The presence of the single O. cf. ovata genotype was confirmed by a PCR assay. Bloom developed when the seawater temperature was decreasing. Nutrient concentrations did not seem to affect bloom dynamics. Toxin analysis performed by high resolution liquid chromatography-mass spectrometry revealed a high total toxin content (up to 75 pg cell(-1)), including putative palytoxin and all the ovatoxins known so far.

Characterization of NW Mediterranean Karlodinium spp. (Dinophyceae) strains using morphological, molecular, chemical, and physiological methodologies

Recurrent fish kills in the Spanish Alfacs Bay (NW Mediterranean) have been detected during winter seasons since 1994, and were attributed to an unarmored, ichthyotoxic, dinoflagellate, initially identified as Gyrodinium corsicum Paulmier, Berland, Billard, & Nezan. Several strains were isolated from the bay and their clonal cultures were compared by combined techniques, including light and electron microscopy, internal transcribed spacer and 5.8S rDNA nucleotide sequencing, and HPLC pigment analyses, together with studies of their photochemical performance, growth rates, and toxicity. Using phylogenetic analyses, all strains were identified as members of the genus Karlodinium, but they were separated into two genetically distinct groups. These groups, identified as Karlodinium veneficum (Ballantine) J. Larsen and K. armiger Bergholtz, Daugbjerg et. Moestrup, were also supported by the other techniques used. Detailed analyses of fine structural characteristics (including plug‐like structures in amphiesma and a possible layer of semi‐opaque material beneath the outer membrane) allowed discrimination of the mentioned two species. Specific differences in pigment patterns coincided with that expected for low‐ (K. veneficum) and high‐light (K. armiger) adapted relatives. The higher photosynthetic efficiency of K. veneficum and the longer reactivation times of the PSII reaction centers observed for K. armiger were in agreement with this hypothesis. The two species differed in toxicity, but the strains used always induced mortality when incubated with bivalves, rotifers, and finfish. Compared with K. armiger, strains of K. veneficum yielded higher cell densities, but had lower growth rates.

Parasitic diatoms inside antarctic sponges.

Antarctic sponges may host large populations of planktonic and benthic diatoms. After settling on the sponge, these diatoms enter its body through pinacocytes (1) and form, there, large mono- or pauci-specific assemblages. Yet the total amount of carbohydrates in the invaded sponge tissue is inversely correlated with that of chlorophyll-a. We suggest, therefore, that endobiont diatoms utilize the products of the metabolism of their host as an energy source. This is the first evidence indicating that an endobiotic autotrophic organism may parasitize its animal host. Moreover, this unusual symbiotic behavior could be a successful strategy that allows the diatom to survive in darkness.

Phylogenetic relationships among the Mediterranean Alexandrium (Dinophyceae) species based on sequences of 5.8S gene and Internal Transcript Spacers of the rRNA operon

A phylogenetic analysis of the genus Alexandrium, including both the most common and rare species from coastal areas of the Mediterranean Sea was carried out. Nucleotide sequences of 5.8 S gene and Internal Transcribed Spacer regions of the rRNA operon were examined and analysed together with isolates of Alexandrium spp. from elsewhere in the world. These rDNA ribosomal markers were useful in delineating the phylogenetic position of species in the genus, as well as in determining relationships among isolates within each species collected from different localities. Results of phylogeographical analyses within the ‘Alexandrium tamarense’ species complex identified three lineages in the Mediterranean Sea: the Mediterranean (ME), Western European (WE) and Temperate Asian (TA) clades. The phylogenetic grouping of the isolates is consistent with the ribotype clades, but not with the morpho-species that constitute the complex. Additional non-toxic isolates were included in the ME clade. The NA (North Atlantic) clade is the fourth group within the ‘Alexandrium tamarense’ species complex identified by phylogenetic analyses. Based on its higher genetic diversity and phylogeographical relationships, it can be hypothesized that the NA clade represents the ancestral group of the ‘Alexandrium tamarense’ species complex. Alexandrium minutum isolates of the NW Mediterranean clustered with strains from Brittany and Australia. Alexandrium minutum constituted a sister clade of A. tamutum, which is another species strongly associated with the Mediterranean area. Another typical Mediterranean species, A. taylori, was placed as a sister clade of A. pseudogoniaulax by the phylogenetic analysis. Finally, the phylogenetic relationships of some Alexandrium morpho-species that were infrequently observed in the Mediterranean Sea have been resolved.