Assessment of microbial plankton diversity as an ecological indicator in the NW Mediterranean coast.

High-throughput sequencing of microbial assemblages has been proposed as an alternative methodology to the traditional ones used in marine monitoring and environmental assessment. Here, we evaluated pico- and nanoplankton diversity as ecological indicators in NW Mediterranean coastal waters by comparing their diversity in samples subjected to varying degrees of continental pressures. Using metabarcoding of the 16S and 18S rRNA genes, we explored whether alphadiversity indices, abundance of Operational Taxonomic Units and taxonomic groups (and their ratios) provide information on the ecological quality of coastal waters. Our results revealed that only eukaryotic diversity metrics and a limited number of prokaryotic and eukaryotic taxa displayed potential in assessing continental influences in our surveyed area, resulting thus in a restrained potential of microbial plankton diversity as an ecological indicator. Therefore, incorporating microbial plankton diversity in environmental assessment could not always result in a significant improvement of current marine monitoring strategies.

Estimation of absolute number of alveolar epithelial type 2 cells in mouse lungs: a comparison between stereology and flow cytometry.

Accurate estimation of the absolute number of a particular cell-type in whole organs is increasingly important in studies on organogenesis, and the remodelling and repair of diseased tissues. The unbiased estimation of the absolute number of cells in an organ is complicated, and design-based stereology remains the method of choice. This has led investigators to explore alternative approaches - such as flow cytometry - as a faster and less labour-intensive replacement for stereology. To address whether flow cytometry might substitute stereology, design-based stereology was compared with microfluorosphere-controlled flow cytometry, for estimation of the absolute number of alveolar epithelial type 2 cells (AEC2) in the lungs of two mouse strains: wild-type C57BL/6J mice and Sftpc-YFP mice. Using design-based stereology, ≈10.7 million and ≈9.0 million AEC2 were estimated in the lungs of wild-type C57BL/6J mice and Sftpc-YFP mice, respectively. Substantially fewer AEC2 were estimated using flow cytometry. In wild-type C57/BL6J mouse lungs, 59% of the AEC2 estimated by design-based stereology were estimated by flow cytometry (≈6.3 million), using intracellular staining for pro-surfactant protein C. Similarly, in Sftpc-YFP mouse lungs, 23% of the AEC2 estimated by design-based stereology were estimated by flow cytometry (≈2.1 million), using yellow fluorescent protein fluorescence. Our data suggest that flow cytometry underestimates AEC2 number, possibly due to impaired recoverability of AEC2 from dissociated lung tissue. These data suggest design-based stereology as the method of choice for the unbiased estimation of the absolute number of cells in an organ. LAY DESCRIPTION: There is much interest in studies on the pathological changes that accompany disease, to be able to count or estimate the number of a particular cell-type in solid tissue, such as an organ. The easiest way to do this is to make liquid suspensions of single cells from solid tissue, and then to count the number of cells of interest, using either a microscope, or automated cell counting (for example, a flow cytometer). Alternatively, solid tissue may be examined microscopically, where the cell-type of interest might also be counted 'by eye' or in an automated manner using software (called planimetry). All of these approaches to counting cells in solid organs come with serious drawbacks, and estimation of the cell number may thus be inaccurate. To overcome this, we have employed a combination of mathematical tools and statistical principles together with microscopy (called 'design-based stereology') that permits the unbiased counting of cells in microscopic fields, which can then be extrapolated to the entire solid tissue volume, to accurately estimate the number of a cell-type of interest in the solid tissue. We have compared this method with the estimation of cell number using a flow cytometer. Our data reveal that flow cytometry appreciably underestimates the total number of cells in solid tissue, where we used the lung as an example of solid tissue, and estimated the number of a unique cell-type in the lung: the alveolar epithelial type 2 cell, to compare stereology with flow cytometry. We believe that flow cytometry underestimates the cell number due to the difficulty of breaking up solid tissue into single cells, and being able to recover all of those single cells for analysis. Our data supports the recommendation to use stereology, not flow cytometry, to accurately estimate the number of a particular cell-type in solid tissue. Accurate estimation of the absolute number of a particular cell-type in whole organs is increasingly important in studies on organogenesis, and the remodelling and repair of diseased tissues. Although estimation of the relative number of cells might be straightforward, unbiased estimation of the absolute number of cells in an organ is complicated, and design-based stereology remains the method of choice. This has led investigators to explore alternative approaches - such as flow cytometry - as a faster and less labour-intensive replacement for stereology. To address whether flow cytometry might substitute stereology, design-based stereology was compared with microfluorosphere-controlled flow cytometry, for estimation of the absolute number of alveolar epithelial type 2 cells (AEC2) in the lungs of two mouse strains: wild-type C57BL/6J mice and Sftpc-YFP mice. Using design-based stereology, ≈10.7 million and ≈9.0 million AEC2 were estimated in the lungs of wild-type C57BL/6J mice and Sftpc-YFP mice, respectively. Substantially fewer AEC2 were estimated using flow cytometry. In wild-type C57/BL6J mouse lungs, 59% of the AEC2 estimated by design-based stereology were estimated by flow cytometry (≈6.3 million), using intracellular staining for pro-surfactant protein C. Similarly, in Sftpc-YFP mouse lungs, 23% of the AEC2 estimated by design-based stereology were estimated by flow cytometry (≈2.1 million), using yellow fluorescent protein fluorescence. Our data suggest that flow cytometry underestimates AEC2 number, possibly due to impaired recoverability of AEC2 from dissociated lung tissue. These data suggest design-based stereology as the method of choice for the unbiased estimation of the absolute number of cells in an organ.

Targeting miR-34a/Pdgfra interactions partially corrects alveologenesis in experimental bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a common complication of preterm birth characterized by arrested lung alveolarization, which generates lungs that are incompetent for effective gas exchange. We report here deregulated expression of miR-34a in a hyperoxia-based mouse model of BPD, where miR-34a expression was markedly increased in platelet-derived growth factor receptor (PDGFR)α-expressing myofibroblasts, a cell type critical for proper lung alveolarization. Global deletion of miR-34a; and inducible, conditional deletion of miR-34a in PDGFRα+ cells afforded partial protection to the developing lung against hyperoxia-induced perturbations to lung architecture. Pdgfra mRNA was identified as the relevant miR-34a target, and using a target site blocker in vivo, the miR-34a/Pdgfra interaction was validated as a causal actor in arrested lung development. An antimiR directed against miR-34a partially restored PDGFRα+ myofibroblast abundance and improved lung alveolarization in newborn mice in an experimental BPD model. We present here the first identification of a pathology-relevant microRNA/mRNA target interaction in aberrant lung alveolarization and highlight the translational potential of targeting the miR-34a/Pdgfra interaction to manage arrested lung development associated with preterm birth.

In situ Occurrence, Prevalence and Dynamics of Parvilucifera Parasitoids during Recurrent Blooms of the Toxic Dinoflagellate Alexandrium minutum.

Dinoflagellate blooms are natural phenomena that often occur in coastal areas, which in addition to their large number of nutrient-rich sites are characterized by highly restricted hydrodynamics within bays, marinas, enclosed beaches, and harbors. In these areas, massive proliferations of dinoflagellates have harmful effects on humans and the ecosystem. However, the high cell density reached during blooms make them vulnerable to parasitic infections. Under laboratory conditions parasitoids are able to exterminate an entire host population. In nature, Parvilucifera parasitoids infect the toxic dinoflagellate Alexandrium minutum during bloom conditions but their prevalence and impact remain unexplored. In this study, we evaluated the in situ occurrence, prevalence, and dynamics of Parvilucifera parasitoids during recurrent blooms of A. minutum in a confined site in the NW Mediterranean Sea as well as the contribution of parasitism to bloom termination. Parvilucifera parasitoids were recurrently detected from 2009 to 2013, during seasonal outbreaks of A. minutum. Parasitic infections in surface waters occurred after the abundance of A. minutum reached 104-105 cells L-1, suggesting a density threshold beyond which Parvilucifera transmission is enhanced and the number of infected cells increases. Moreover, host and parasitoid abundances were not in phase. Instead, there was a lag between maximum A. minutum and Parvilucifera densities, indicative of a delayed density-dependent response of the parasitoid to host abundances, similar to the temporal dynamics of predator-prey interactions. The highest parasitoid prevalence was reached after a peak in host abundance and coincided with the decay phase of the bloom, when a maximum of 38% of the A. minutum population was infected. According to our estimates, Parvilucifera infections accounted for 5-18% of the total observed A. minutum mortality, which suggested that the contribution of parasitism to bloom termination is similar to that of other biological factors, such as encystment and grazing.

Morphological and molecular characterization of Bysmatrum subsalsum (Dinophyceae) from the western Mediterranean Sea reveals the existence of cryptic species.

Bysmatrum subsalsum is a cosmopolitan dinoflagellate species that inhabits marine and transitional habitats. Despite its wide distribution, information on the morphological variability, phylogeny and ecology of B. subsalsum is scarce. In this study, we provide morphological and molecular data on B. subsalsum strains and wild cells from different locations in the Mediterranean Basin. The dynamics of cell abundances and the associated environmental conditions during a field bloom are also described. Genetic sequences of B. subsalsum obtained in this study showed large intraspecific differences, clustering in two well-differentiated clades. Despite a certain degree of variation with respect to cell size, apical pore complex (APC) morphology and size, and cingulum displacement, cells from the two clades showed similar morphological traits. These findings indicated the occurrence of cryptic species. Comparisons of the morphology of our B. subsalsum specimens with the few descriptions available in the literature revealed larger than previously known intraspecific morphological variability. Phylogenetic trees inferred from the concatenated SSU, 5.8S-ITS, and LSU rRNA and the individual 5.8S-ITS regions suggested the inclusion of Bysmatrum in the Peridiniales and a close phylogenetic relationship with Peridinium sensu stricto. However, the low statistical support prevented the assignment of Bysmatrum to a particular family of Peridiniales. Ecological data obtained from a bloom in La Pletera salt marshes (Catalan Coast, Spain) suggested the species reaches high cell abundances at water temperatures >20°C and salinity levels >30. Our results add new information regarding the morphology, phylogeny, and ecology of B. subsalsum.

Standardisation of oxygen exposure in the development of mouse models for bronchopulmonary dysplasia.

Progress in developing new therapies for bronchopulmonary dysplasia (BPD) is sometimes complicated by the lack of a standardised animal model. Our objective was to develop a robust hyperoxia-based mouse model of BPD that recapitulated the pathological perturbations to lung structure noted in infants with BPD. Newborn mouse pups were exposed to a varying fraction of oxygen in the inspired air (FiO2) and a varying window of hyperoxia exposure, after which lung structure was assessed by design-based stereology with systemic uniform random sampling. The efficacy of a candidate therapeutic intervention using parenteral nutrition was evaluated to demonstrate the utility of the standardised BPD model for drug discovery. An FiO2 of 0.85 for the first 14 days of life decreased total alveoli number and concomitantly increased alveolar septal wall thickness, which are two key histopathological characteristics of BPD. A reduction in FiO2 to 0.60 or 0.40 also caused a decrease in the total alveoli number, but the septal wall thickness was not impacted. Neither a decreasing oxygen gradient (from FiO2 0.85 to 0.21 over the first 14 days of life) nor an oscillation in FiO2 (between 0.85 and 0.40 on a 24 h:24 h cycle) had an appreciable impact on lung development. The risk of missing beneficial effects of therapeutic interventions at FiO2 0.85, using parenteral nutrition as an intervention in the model, was also noted, highlighting the utility of lower FiO2 in selected studies, and underscoring the need to tailor the model employed to the experimental intervention. Thus, a state-of-the-art BPD animal model that recapitulates the two histopathological hallmark perturbations to lung architecture associated with BPD is described. The model presented here, where injurious stimuli have been systematically evaluated, provides a most promising approach for the development of new strategies to drive postnatal lung maturation in affected infants.

Tamoxifen dosing for Cre-mediated recombination in experimental bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is the most common complication of preterm birth characterized by blunted post-natal lung development. BPD can be modelled in mice by exposure of newborn mouse pups to elevated oxygen levels. Little is known about the mechanisms of perturbed lung development associated with BPD. The advent of transgenic mice, where genetic rearrangements can be induced in particular cell-types at particular time-points during organogenesis, have great potential to explore the pathogenic mechanisms at play during arrested lung development. Many inducible, conditional transgenic technologies available rely on the application of the estrogen-receptor modulator, tamoxifen. While tamoxifen is well-tolerated and has been widely employed in adult mice, or in healthy developing mice; tamoxifen is not well-tolerated in combination with hyperoxia, in the most widely-used mouse model of BPD. To address this, we set out to establish a safe and effective tamoxifen dosing regimen that can be used in newborn mouse pups subjected to injurious stimuli, such as exposure to elevated levels of environmental oxygen. Our data reveal that a single intraperitoneal dose of tamoxifen of 0.2 mg applied to newborn mouse pups in 10 µl Miglyol vehicle was adequate to successfully drive Cre recombinase-mediated genome rearrangements by the fifth day of life, in a murine model of BPD. The number of recombined cells was comparable to that observed in regular tamoxifen administration protocols. These findings will be useful to investigators where tamoxifen dosing is problematic in the background of injurious stimuli and mouse models of human and veterinary disease.

Divergent fibroblast growth factor signaling pathways in lung fibroblast subsets: where do we go from here?

Lung fibroblasts play a key role in postnatal lung development, namely, the formation of the alveolar gas exchange units, through the process of secondary septation. Although evidence initially highlighted roles for fibroblasts in the production and remodeling of the lung extracellular matrix, more recent studies have described the presence of different fibroblast subsets in the developing lung. These subsets include myofibroblasts and lipofibroblasts and their precursors. These cells are believed to play different roles in alveologenesis and are localized to different regions of the developing septa. The precise roles played by these different fibroblast subsets remain unclear. Understanding the signaling pathways that control the discrete functions of these fibroblast subsets would help to clarify the roles and the regulation of lung fibroblasts during lung development. Here, we critically evaluate a recent report that described divergent fibroblast growth factor (FGF) signaling pathways in two different subsets of lung fibroblasts that express different levels of green fluorescent protein (GFP) driven by the platelet-derived growth factor receptor-α promoter. The GFP expression was used as a surrogate for lipofibroblasts (GFP(low)) and myofibroblasts (GFP(high)). It was suggested that Fgf10/Fgf1 and Fgf18/Fgfr3 autocrine pathways may be operative in GFP(low) and GFP(high) cells, respectively, and that these pathways might regulate the proliferation and migration of different fibroblast subsets during alveologenesis. These observations lay important groundwork for the further exploration of FGF function during normal lung development, as well as in aberrant lung development associated with bronchopulmonary dysplasia.

Collagen and elastin cross-linking is altered during aberrant late lung development associated with hyperoxia.

Maturation of the lung extracellular matrix (ECM) plays an important role in the formation of alveolar gas exchange units. A key step in ECM maturation is cross-linking of collagen and elastin, which imparts stability and functionality to the ECM. During aberrant late lung development in bronchopulmonary dysplasia (BPD) patients and animal models of BPD, alveolarization is blocked, and the function of ECM cross-linking enzymes is deregulated, suggesting that perturbed ECM cross-linking may impact alveolarization. In a hyperoxia (85% O2)-based mouse model of BPD, blunted alveolarization was accompanied by alterations to lung collagen and elastin levels and cross-linking. Total collagen levels were increased (by 63%). The abundance of dihydroxylysinonorleucine collagen cross-links and the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio were increased by 11 and 18%, respectively, suggestive of a profibrotic state. In contrast, insoluble elastin levels and the abundance of the elastin cross-links desmosine and isodesmosine in insoluble elastin were decreased by 35, 30, and 21%, respectively. The lung collagen-to-elastin ratio was threefold increased. Treatment of hyperoxia-exposed newborn mice with the lysyl oxidase inhibitor ß-aminopropionitrile partially restored normal collagen levels, normalized the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio, partially normalized desmosine and isodesmosine cross-links in insoluble elastin, and partially restored elastin foci structure in the developing septa. However, ß-aminopropionitrile administration concomitant with hyperoxia exposure did not improve alveolarization, evident from unchanged alveolar surface area and alveoli number, and worsened septal thickening (increased by 12%). These data demonstrate that collagen and elastin cross-linking are perturbed during the arrested alveolarization of developing mouse lungs exposed to hyperoxia.

Diversity and Phylogeny of Gymnodiniales (Dinophyceae) from the NW Mediterranean Sea Revealed by a Morphological and Molecular Approach.

The diversity and phylogeny of dinoflagellates belonging to the Gymnodiniales were studied during a 3-year period at several coastal stations along the Catalan coast (NW Mediterranean) by combining analyses of their morphological features with rDNA sequencing. This approach resulted in the detection of 59 different morphospecies, 13 of which were observed for the first time in the Mediterranean Sea. Fifteen of the detected species were HAB producers; four represented novel detections on the Catalan coast and two in the Mediterranean Sea. Partial rDNA sequences were obtained for 50 different morphospecies, including novel LSU rDNA sequences for 27 species, highlighting the current scarcity of molecular information for this group of dinoflagellates. The combination of morphology and genetics allowed the first determinations of the phylogenetic position of several genera, i.e., Torodinium and many Gyrodinium and Warnowiacean species. The results also suggested that among the specimens belonging to the genera Gymnodinium, Apicoporus, and Cochlodinium were those representing as yet undescribed species. Furthermore, the phylogenetic data suggested taxonomic incongruences for some species, i.e., Gyrodinium undulans and Gymnodinium agaricoides. Although a species complex related to G. spirale was detected, the partial LSU rDNA sequences lacked sufficient resolution to discriminate between various other Gyrodinium morphospecies.

Systemic hydrogen sulfide administration partially restores normal alveolarization in an experimental animal model of bronchopulmonary dysplasia.

Arrested alveolarization is the pathological hallmark of bronchopulmonary dysplasia (BPD), a complication of premature birth. Here, the impact of systemic application of hydrogen sulfide (H2S) on postnatal alveolarization was assessed in a mouse BPD model. Exposure of newborn mice to 85% O2 for 10 days reduced the total lung alveoli number by 56% and increased alveolar septal wall thickness by 29%, as assessed by state-of-the-art stereological analysis. Systemic application of H2S via the slow-release H2S donor GYY4137 for 10 days resulted in pronounced improvement in lung alveolarization in pups breathing 85% O2, compared with vehicle-treated littermates. Although without impact on lung oxidative status, systemic H2S blunted leukocyte infiltration into alveolar air spaces provoked by hyperoxia, and restored normal lung interleukin 10 levels that were otherwise depressed by 85% O2. Treatment of primary mouse alveolar type II (ATII) cells with the rapid-release H2S donor NaHS had no impact on cell viability; however, NaHS promoted ATII cell migration. Although exposure of ATII cells to 85% O2 caused dramatic changes in mRNA expression, exposure to either GYY4137 or NaHS had no impact on ATII cell mRNA expression, as assessed by microarray, suggesting that the effects observed were independent of changes in gene expression. The impact of NaHS on ATII cell migration was attenuated by glibenclamide, implicating ion channels, and was accompanied by activation of Akt, hinting at two possible mechanisms of H2S action. These data support further investigation of H2S as a candidate interventional strategy to limit the arrested alveolarization associated with BPD.

Polykrikos tanit sp. nov., a new mixotrophic unarmoured pseudocolonial dinoflagellate from the NW Mediterranean Sea.

Pigmented pseudocolonies initially identified as Polykrikos hartmannii Zimmermann were detected at several locations of the Catalan coast (NW Mediterranean Sea) in April-June of 2012 and April-May of 2013. To further explore the several remarkable morphological discrepancies between these organisms and P. hartmannii, we carried out a detailed morphological study and used single-cell PCR to obtain partial LSU and SSU rDNA sequences. The resulting phylogenies showed that our isolates occupy a basal position within the Polykrikos clade, close to P. hartmannii, but do not correspond to any described polykrikoid species. P. barnegatensis Martin is controversially considered to be synonymous with P. hartmannii. The organisms studied in this work were similar to P. barnegatensis but showed significant morphological differences with its original description such as the torsion of the pseudocolony, more pronounced overhanging of the cingula, stepped fusion border of the zooids, and number and shape of nuclei. Consequently, we propose that the isolates constitute a new species, which we named Polykrikos tanit sp. nov. The observed characters, pigmented, same number of zooids and nuclei, sulci not fused, and its phylogeny suggest that the species is an early evolutionary Polykrikos species.

Recent advances in late lung development and the pathogenesis of bronchopulmonary dysplasia.

In contrast to early lung development, a process exemplified by the branching of the developing airways, the later development of the immature lung remains very poorly understood. A key event in late lung development is secondary septation, in which secondary septa arise from primary septa, creating a greater number of alveoli of a smaller size, which dramatically expands the surface area over which gas exchange can take place. Secondary septation, together with architectural changes to the vascular structure of the lung that minimize the distance between the inspired air and the blood, are the objectives of late lung development. The process of late lung development is disturbed in bronchopulmonary dysplasia (BPD), a disease of prematurely born infants in which the structural development of the alveoli is blunted as a consequence of inflammation, volutrauma, and oxygen toxicity. This review aims to highlight notable recent developments in our understanding of late lung development and the pathogenesis of BPD.

A new clade, based on partial LSU rDNA sequences, of unarmoured dinoflagellates.

The order Gymnodiniales comprises unarmoured dinoflagellates. However, the lack of sequences hindered determining the phylogenetic positions and systematic relationships of several gymnodinioid taxa. In this study, a monophyletic clade was defined for the species Ceratoperidinium margalefii Loeblich III, Gyrodinium falcatum Kofoid & Swezy, three Cochlodinium species, and two Gymnodinium-like dinoflagellates. Despite their substantial morphotypic differentiation, Cochlodinium cf. helix, G. falcatum and 'Gymnodinium' sp. 1 share a common shape of the acrobase. The phylogenetic data led to the following conclusions: (1) C. margalefii is closely related to several unarmoured dinoflagellates. Its sulcus shape has been observed for the first time. (2) G. falcatum was erroneously assigned to the genus Gyrodinium and is transferred to Ceratoperidinium (C. falcatum (Kofoid & Swezy) Reñé & de Salas comb. nov.). (3) The genus Cochlodinium is polyphyletic and thus artificial; our data support its separation into three different genera. (4) The two Gymnodinium-like species could not be morphologically or phylogenetically related to any other gymnodinioid species sequenced to date. While not all studied species have been definitively transferred to the correct genus, our study is a step forward in the classification of inconspicuous unarmoured dinoflagellates. The family Ceratoperidiniaeceae and the genus Ceratoperidinium are emended.

Results of implementation of a fast track pathway for diagnosis of colorectal cancer.

INTRODUCTION: fast track pathways for diagnosis of cancer intend to decrease delays in diagnosis and treatment of cancer. It is recommended to initiate treatment in a period no longer than 30 days since admission in these circuits. AIMS: to know the characteristics and fluency of our Fast Track Diagnostic Pathway (FTDP) for Colorectal Cancer (CRC), with special attention to those patients selected for surgical treatment as a first choice. MATERIAL AND METHOD: all patients who entered the FTDP for CRC during a period of 2 years (2008-2009) were analyzed as well as the rest of patients also diagnosed with CRC but never seen in the FTDP. RESULTS: of the 316 patients referred to the FTDP only 78 (24.7%) were diagnosed as having some kind of cancer derived from the digestive system. At the end 61 patients (19.3%) were diagnosed with CCR. The time interval from entry into the FTDP to the first hospital visit was 3 days (range 1-8), and the interval until colonoscopy was performed was 11.5 days (range 1-41). Fourteen (41.1%) of those patients chosen for surgery were operated on in a period lesser than 30 days while 28 patients (82.3%) underwent surgery before day 45 since admission into the circuit. CONCLUSIONS: though the functioning of the FTDP is acceptable, any increase in number of patients can generate delays. For this reason it is advisable to have a team to assure a good functioning of the FTDP. A proper follow-up of the whole process will possibly avoid unnecessary delays and it will improve coordination of the different phases of the fast track pathway and treatment. As the diagnostic outcome is poor it is mandatory to implement alternatives programs like screening of asymptomatic population, allowing an early detection of this condition.

Quantitative PCR coupled with melt curve analysis for detection of selected pseudo-nitzschia spp. (Bacillariophyceae) from the Northwestern Mediterranean sea.

The frequency and intensity of Pseudo-nitzschia spp. blooms along the coast of Catalonia have been increasing over the past 20 years. As species from this genus that are documented as toxigenic have been found in local waters, with both toxic and nontoxic species cooccurring in the same bloom, there is a need to develop management tools for discriminating the difference. Currently, differentiation of toxic and nontoxic species requires time-consuming electron microscopy to distinguish taxonomic features that would allow identification as to species, and cryptic species can still remain misidentified. In this study, cells of Pseudo-nitzschia from clonal cultures isolated from seawater were characterized to their species identity using scanning electron microscopy, and subsamples of each culture were used to create an internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 ribosomal DNA database for development of species-specific quantitative PCR (qPCR) assays. Once developed, these qPCR assays were applied to field samples collected over a 2-year period in Alfaques Bay in the northwestern Mediterranean Sea to evaluate the possibility of a comprehensive surveillance for all Pseudo-nitzschia spp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. Total Pseudo-nitzschia cell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eight Pseudo-nitzschia species-specific qPCR assays using duplicate samples. Species-specific qPCR followed by melt curve analysis allowed differentiation of amplicons and identification of false positives, and results correlated well with the total Pseudo-nitzschia cell counts from optical microscopy.

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.

Use of the dinoflagellate Karlodinium veneficum as a sustainable source of biodiesel production.

Microalgae are microscopic heterotrophic-autotrophic photosynthesizing organisms with enormous potential as a source of biofuel. Dinoflagellates, a class of microalgae, contain large amounts of high-quality lipids, the principal component of fatty acid methyl esters. The biotic characteristics of the dinoflagellate species Karlodinium veneficum include a growth rate of 0.14 day(-1), a wet biomass of 16.4 g/L, a growth period of approximately 30 days, and an approximate 97% increase in fatty acid content during the transition from exponential phase to stationary phase. These parameters make K. veneficum a suitable choice as a bioresource for biodiesel production. Similarly, two other species were also determined to be appropriate for biodiesel production: the Dinophyceae Alexandrium andersoni and the Raphidophyte Heterosigma akashiwo.

HOMOTHALLIC AUXOSPORULATION IN PSEUDO-NITZSCHIA BRASILIANA (BACILLARIOPHYTA)(1).

Most pennate diatoms are allogamous, and various types of mating systems have been described. In Pseudo-nitzschia, reproductive stages have been identified in some species, and it is generally accepted that the genus is mainly heterothallic. Here we report homothallic auxosporulation of Pseudo-nitzschia brasiliana Lundholm, Hasle et G. A. Fryxell. To our knowledge, this is the first verified description of homothallic sexual reproduction in the genus. Auxospore formation was observed in all 16 subclones derived from three initial clonal cultures of P. brasiliana. Pairing was followed by production of two gametes per gametangium, which fused to give two zygotes. Each zygote (early auxospore) was initially spherical and adhered to one girdle band of the parental frustule. The two auxospores tended to expand parallel to each other and perpendicular to the parental frustule. Elongation was synchronous, slightly asynchronous, or totally asynchronous. The entire process of sexual reproduction, from gamete formation to the appearance of the initial vegetative cells, took 2-4 d. The occurrence of sex in a homothallic species seems an advantageous life strategy for this species in that any encounter between cells of the right size class is potentially sexual.

Description, host-specificity, and strain selectivity of the dinoflagellate parasite Parvilucifera sinerae sp. nov. (Perkinsozoa).

A new species of parasite, Parvilucifera sinerae sp. nov., isolated from a bloom of the toxic dinoflagellate Alexandrium minutum in the harbor of Arenys de Mar (Mediterranean Sea, Spain), is described. This species is morphologically, behaviourally, and genetically (18S rDNA sequence) different from Parvilucifera infectans, until now the only species of the genus Parvilucifera to be genetically analyzed. Sequence analysis of the 18S ribosomal DNA supported P. sinerae as a new species placed within the Perkinsozoa and close to P. infectans. Data on the seasonal occurrence of P. sinerae, its infective rates in natural and laboratory cultures, and intra-species strain-specific resistance are presented. Life-cycle studies in field samples showed that the dinoflagellate resting zygote (resting cyst) was resistant to infection, but the mobile zygote (planozygote) or pellicle stage (temporary cyst) became infected. The effects of light and salinity levels on the growth of P. sinerae were examined, and the results showed that low salinity levels promote both sporangial germination and higher rates of infection. Our findings on this newly described parasite point to a complex host-parasite interaction and provide valuable information that leads to a reconsideration of the biological strategy to control dinoflagellate blooms by means of intentional parasitic infections.