Temperature-dependent growth and sexuality of the ciguatoxin producer dinoflagellate Gambierdiscus spp. in cultures established from the Canary Islands.

Benthic dinoflagellates of the genus Gambierdiscus produce ciguatoxins, compounds that when metabolized in fish and consumed by humans cause ciguatera poisoning (CP). This syndrome, which is widespread in tropical and subtropical regions, has recently been reported also in subtropical-temperate latitudes such as the Canary Islands where CP events have been regularly detected since 2004. This study examined the effect of temperature on the growth of Gambierdiscus isolated from Canary waters: G. australes, G. caribaeus, G. carolinianus, G. excentricus, and G. silvae. From the temperature vs. growth curves, the maximum growth (µm), optimum temperature range for growth (Topt), and the temperature yielding maximum growth (Tm) were estimated for each species. The results revealed temperature-dependent differences in the growth parameters. G. caribaeus had the highest Tm and Topt, followed by G. australes, G. carolinianus, G. silvae, and G excentricus. G. australes tolerated the widest range of temperatures (from 15 °C to 29 °C), which may explain its broader geographic distribution, both worldwide and across the Canary archipelago. Neither G. excentricus nor G. silvae survived at 29 °C whereas G. caribaeus reached mean growth rates (± standard deviation) up to 0.19 ± 0.01 div.day-1 at that temperature, followed by G. australes (0.16 ± 0.01 div.day-1) and G. carolinianus (0.14 ± 0.04 div.day-1). G. caribaeus showed no measurable growth at 19°C, whereas G. excentricus and G. silvae along with G. australes appeared as the species better adapted to lower temperatures. In an intraspecific variability study of 12 strains of G. australes, the mean (± standard deviation) of µm and Tm were 0.17 ± 0.01 div.day-1 and 27.7 ± 0.5 °C, respectively. An analysis of the shapes and position of the cell nuclei at the different temperatures showed that nuclei characteristic of vegetative cells appeared mainly at 26 °C but extreme temperatures resulted in nuclei with a more variable morphology. The presence of putative zygotes at extreme temperatures (17 °C, 19 °C and 29 °C) suggests that sexual reproduction is promoted as an adaptive strategy which could play an important role in the expansion of geographic distribution of Gambierdiscus species.

Morphological and phylogenetic data do not support the split of Alexandrium into four genera.

A recently published study analyzed the phylogenetic relationship between the genera Centrodinium and Alexandrium, confirming an earlier publication showing the genus Alexandrium as paraphyletic. This most recent manuscript retained the genus Alexandrium, introduced a new genus Episemicolon, resurrected two genera, Gessnerium and Protogonyaulax, and stated that: "The polyphyly [sic] of Alexandrium is solved with the split into four genera". However, these reintroduced taxa were not based on monophyletic groups. Therefore this work, if accepted, would result in replacing a single paraphyletic taxon with several non-monophyletic ones. The morphological data presented for genus characterization also do not convincingly support taxa delimitations. The combination of weak molecular phylogenetics and the lack of diagnostic traits (i.e., autapomorphies) render the applicability of the concept of limited use. The proposal to split the genus Alexandrium on the basis of our current knowledge is rejected herein. The aim here is not to present an alternative analysis and revision, but to maintain Alexandrium. A better constructed and more phylogenetically accurate revision can and should wait until more complete evidence becomes available and there is a strong reason to revise the genus Alexandrium. The reasons are explained in detail by a review of the available molecular and morphological data for species of the genera Alexandrium and Centrodinium. In addition, cyst morphology and chemotaxonomy are discussed, and the need for integrative taxonomy is highlighted.

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.

Life histories of microalgal species causing harmful blooms: Haploids, diploids and the relevance of benthic stages.

In coastal and offshore waters, Harmful Algal Blooms (HABs) currently threaten the well-being of coastal countries. These events, which can be localized or involve wide-ranging areas, pose risks to human health, marine ecosystems, and economic resources, such as tourism, fisheries, and aquaculture. Dynamics of HABs vary from one site to another, depending on the hydrographic and ecological conditions. The challenge in investigating HABs is that they are caused by organisms from multiple algal classes, each with its own unique features, including different life histories. The complete algal life cycle has been determined in <1% of the described species, although elucidation of the life cycles of bloom-forming species is essential in developing preventative measures. The knowledge obtained thus far has confirmed the complexity of the algal life cycle, which is composed of discrete life stages whose morphology, ecological niche (plankton/benthos), function, and lifespan vary. The factors that trigger transitions between the different stages in nature are mostly unknown, but it is clear that an understanding of this process provides the key to effectively forecasting bloom recurrence, maintenance, and decline. Planktonic stages constitute an ephemeral phase of the life cycle of most species whereas resistant, benthic stages enable a species to withstand adverse conditions for prolonged periods, thus providing dormant reservoirs for eventual blooms and facilitating organismal dispersal. Here we review current knowledge of the life cycle strategies of major groups of HAB producers in marine and brackish waters. Rather than providing a comprehensive discussion, the objective was to highlight several of the research milestones that have changed our understanding of the plasticity and frequency of the different life cycle stages as well as the transitions between them. We also discuss the relevance of benthic and planktonic forms and their implications for HAB dynamics.

Life-cycle, ultrastructure, and phylogeny of Parvilucifera corolla sp. nov. (Alveolata, Perkinsozoa), a parasitoid of dinoflagellates.

Recent studies of marine protists have revealed parasites to be key components of marine communities. Here we describe a new species of the parasitoid genus Parvilucifera that was observed infecting the dinoflagellate Durinskia baltica in salt marshes of the Catalan coast (NW Mediterranean). In parallel, the same species was detected after the incubation of seawater from the Canary Islands (Lanzarote, NE Atlantic). The successful isolation of strains from both localities allowed description of the life cycle, ultrastructure, and phylogeny of the species. Its infection mechanism consists of a free-living zoospore that penetrates a dinoflagellate cell. The resulting trophont gradually degrades the dinoflagellate cytoplasm while growing in size. Once the host is consumed, schizogony of the parasitoid yields a sporocyte. After cytokinesis is complete, the newly formed zoospores are released into the environment and are ready to infect new host cells. A distinguishing feature of the species is the radial arrangement of its zoospores around the central area of the sporocyte during their formation. The species shows a close morphological similarity with other species of the genus, including P. infectans, P. sinerae, and P. rostrata.

"Canary Islands (NE Atlantic) as a biodiversity 'hotspot' of Gambierdiscus: Implications for future trends of ciguatera in the area".

In the present study the geographical distribution, abundance and composition of Gambierdiscus was described over a 600km longitudinal scale in the Canary Islands. Samples for cell counts, isolation and identification of Gambierdiscus were obtained from five islands (El Hierro, Tenerife, Gran Canaria, Fuerteventura and Lanzarote). Average densities of Gambierdiscus spp. between 0 and 2200cellsg-1 blot dry weight of macrophyte were recorded. Morphological (light microscopy and SEM techniques) and molecular analyses (LSU and SSU rDNA sequencing of cultures and single cells from the field) of Gambierdiscus was performed. Five Gambierdiscus species (G. australes, G. caribaeus, G. carolinianus, G. excentricus and G. silvae), together with a new putative species (Gambierdiscus ribotype 3) were identified. These results suggest that some cases of CFP in the region could be associated with the accumulation of ciguatoxins in the marine food web acquired from local populations of Gambierdiscus. This unexpected high diversity of Gambierdiscus species in an area which a priori is not under risk of ciguatera, hints at an ancient settlement of Gambierdiscus populations, likely favored by warmer climate conditions in the Miocene Epoch (when oldest current Canary Islands were created), in contrast with cooler present ones. Currently, warming trends associated with climate change could contribute to extend favorable environmental conditions in the area for Gambierdiscus growth especially during winter months.

Nuclear and cell morphological changes during the cell cycle and growth of the toxic dinoflagellate Alexandrium minutum.

Elucidation of the cell cycle of dinoflagellates is essential to understand the processes leading to their massive proliferations, known as harmful algal blooms. In this study, we used imaging flow cytometry (IFC) to monitor the changes in DNA content and nuclear and cell morphology that occur during clonal growth of the toxic species Alexandrium minutum Halim. Our results indicate that the population was in S phase (C→2C DNA content) during the light period, whereas haploid cells with a C DNA content peaked only during a short interval of the dark period. The timing of the phases, identified based on the nuclear morphology and cytoplasmic-to-nuclear (CNR) ratio of the cells, suggests that the length of the G2/M phase is regulated by nutrient levels whereas the beginning of S phase is clock controlled. In addition we found that up to 7% of individual cells achieved a DNA content higher than 2C, indicative of either zygote formation and replication (homothallism), or of double-haploid cells able to divide (polyploid forms). Cells belonging to different cell cycle phases (G1-S-G2/M) could be readily discriminated based on nuclear size. Our study provides evidence of cell-cycle plasticity during clonal growth and unambiguously characterizes the cell-cycle phases of this dinoflagellate species.

Towards an Ecological Understanding of Dinoflagellate Cyst Functions.

The life cycle of many dinoflagellates includes at least one nonflagellated benthic stage (cyst). In the literature, the different types of dinoflagellate cysts are mainly defined based on morphological (number and type of layers in the cell wall) and functional (long- or short-term endurance) differences. These characteristics were initially thought to clearly distinguish pellicle (thin-walled) cysts from resting (double-walled) dinoflagellate cysts. The former were considered short-term (temporal) and the latter long-term (resting) cysts. However, during the last two decades further knowledge has highlighted the great intricacy of dinoflagellate life histories, the ecological significance of cyst stages, and the need to clarify the functional and morphological complexities of the different cyst types. Here we review and, when necessary, redefine the concepts of resting and pellicle cysts, examining both their structural and their functional characteristics in the context of the life cycle strategies of several dinoflagellate species.

Ribosomal DNA organization patterns within the dinoflagellate genus Alexandrium as revealed by FISH: life cycle and evolutionary implications.

Dinoflagellates are a group of protists whose genome differs from that of other eukaryotes in terms of size (contains up to 250pg per haploid cell), base composition, chromosomal organization, and gene expression. But rDNA gene mapping of the active nucleolus in this unusual eukaryotic genome has not been carried out thus far. Here we used FISH in dinoflagellate species belonging to the genus Alexandrium (genome sizes ranging from 21 to 170 pg of DNA per haploid genome) to localize the sequences encoding the 18S, 5.8S, and 28S rRNA genes. The results can be summarized as follows: 1) Each dinoflagellate cell contains only one active nucleolus, with no hybridization signals outside it. However, the rDNA organization varies among species, from repetitive clusters forming discrete nuclear organizer regions (NORs) in some to specialized "ribosomal chromosomes" in other species. The latter chromosomes, never reported before in other eukaryotes, are mainly formed by rDNA genes and appeared in the species with the highest DNA content. 2) Dinoflagellate chromosomes are first characterized by several eukaryotic features, such as structural differentiation (centromere-like constrictions), size differences (dot chromosomes), and SAT (satellite) chromosomes. 3) NOR patterns prove to be useful in discriminating between cryptic species and life cycle stages in protists.

Patterns of post-glacial genetic differentiation in marginal populations of a marine microalga.

This study investigates the genetic structure of an eukaryotic microorganism, the toxic dinoflagellate Alexandrium ostenfeldii, from the Baltic Sea, a geologically young and ecologically marginal brackish water estuary which is predicted to support evolution of distinct, genetically impoverished lineages of marine macroorganisms. Analyses of the internal transcribed spacer (ITS) sequences and Amplified Fragment Length Polymorphism (AFLP) of 84 A. ostenfeldii isolates from five different Baltic locations and multiple external sites revealed that Baltic A. ostenfeldii is phylogenetically differentiated from other lineages of the species and micro-geographically fragmented within the Baltic Sea. Significant genetic differentiation (F(ST)) between northern and southern locations was correlated to geographical distance. However, instead of discrete genetic units or continuous genetic differentiation, the analysis of population structure suggests a complex and partially hierarchic pattern of genetic differentiation. The observed pattern suggests that initial colonization was followed by local differentiation and varying degrees of dispersal, most likely depending on local habitat conditions and prevailing current systems separating the Baltic Sea populations. Local subpopulations generally exhibited low levels of overall gene diversity. Association analysis suggests predominately asexual reproduction most likely accompanied by frequency shifts of clonal lineages during planktonic growth. Our results indicate that the general pattern of genetic differentiation and reduced genetic diversity of Baltic populations found in large organisms also applies to microscopic eukaryotic organisms.

INTERACTIVE EFFECTS OF SALINITY AND TEMPERATURE ON PLANOZYGOTE AND CYST FORMATION OF ALEXANDRIUM MINUTUM (DINOPHYCEAE) IN CULTURE(1).

The factors regulating dinoflagellate life-cycle transitions are poorly understood. However, their identification is essential to unravel the causes promoting the outbreaks of harmful algal blooms (HABs) because these blooms are often associated with the formation and germination of sexual cysts. Nevertheless, there is a lack of knowledge on the factors regulating planozygote-cyst transitions in dinoflagellates due to the difficulties of differentiating planozygotes from vegetative stages. In the present study, two different approaches were used to clarify the relevance of environmental factors on planozygote and cyst formation of the toxic dinoflagellate Alexandrium minutum Halim. First, the effects of changes in initial phosphate (P) and nitrate (N) concentrations in the medium on the percentage of planozygotes formed were examined using flow cytometry. Second, two factorial designs were used to determine how salinity (S), temperature (T), and the density of the initial cell inoculum (I) affect planozygote and resting-cyst formation. These experiments led to the following conclusions: 1. Low P/N ratios seem to induce gamete expression because the percentage of planozygotes recorded in the absence of added phosphate (-P) was significantly higher than that obtained in the absence of added nitrogen (-N), or when the concentrations of both nitrogen and phosphate were 20 times lower (N/20 + P/20). 2. Salinity (S) and temperature (T) strongly affected both planozygote and cyst formation, as sexuality in the population increased significantly as salinity decreased and temperatures increased. S, T combinations that resulted in no significant cyst formation were, however, favorable for vegetative growth, ruling out the possibility of negative effects on cell physiology. 3. The initial cell density is thought to be important for sexual cyst formation by determining the chances of gamete contact. However, the inoculum concentrations tested did not explain either planozygote formation or the appearance of resting cysts.

The life history and cell cycle of Kryptoperidinium foliaceum, a dinoflagellate with two eukaryotic nuclei.

Kryptoperidinium foliaceum is a binucleate dinoflagellate that contains an endosymbiont nucleus of diatom origin. However, it is unknown whether the binucleate condition is permanent or not and how the diatom nucleus behaves during the life history processes. In this sense, it is also unknown if there is a sexual cycle or a resting stage during the life history of this species, two key aspects necessary to understand the life history strategy of this dinoflagellate. To answer these questions, life history and cell cycle studies were performed with the following results: (i) Kryptoperidinium foliaceum has a sexual cycle and in the dinoflagellate strains studied, the binucleate condition is permanent. Sexuality in the host was confirmed by the presence of fusing gamete pairs and planozygotes in clonal cultures (revealing homothallism), but signs of meiosis in the endosymbiont were not observed. The endosymbiont nucleus likely fuses first, because fusing gamete pairs were found to have two dinoflagellate nuclei but only one endosymbiont nucleus. After complete gamete fusion, the planozygotes had apparently normal endosymbiont and dinoflagellate nuclei. (ii) Asexual division studies using flow cytometry showed that the S phase in the endosymbiont (diatom) nucleus starts 6-8h later than in the host nucleus, but there was no evidence of mitosis in the former. (iii) Sexual and asexual cysts were formed in culture. Neither cysts from natural samples nor those formed in culture exhibited a dormancy period before germination.

CELL CYCLE AND CELL MORTALITY OF ALEXANDRIUM MINUTUM (DINOPHYCEAE) UNDER SMALL-SCALE TURBULENCE CONDITIONS(1).

Decreased net population growth rates and cellular abundances have been observed in dinoflagellate species exposed to small-scale turbulence. Here, we investigated whether these effects were caused by alterations in the cell cycle and/or by cell mortality and, in turn, whether these two mechanisms depended on the duration of exposure to turbulence. The study was conducted on the toxic dinoflagellate Alexandrium minutum Halim, with the same experimental design and setup used in previous studies to allow direct comparison among results. A combination of microscopy and Coulter Counter measurements allowed us to detect cell mortality, based on the biovolume of broken cells and thecae. The turbulence applied during the exponential growth phase caused an immediate transitory arrest in the G2/M phase, but significant mortality did not occur. This finding suggests that high intensities of small-scale turbulence can alter the cell division, likely affecting the correct chromosome segregation during the dinomitosis. When shaking persisted for >4 d, mortality signals and presence of anomalously swollen cells appeared, hinting at the activation of mechanisms that induce programmed cell death. Our study suggests that the sensitivity of dinoflagellates to turbulence may drive these organisms to find the most favorable (calm) conditions to complete their division cycle.