Towards an optimal sampling strategy for Alexandrium catenella (Dinophyceae) benthic resting cysts

The study proposes methodological developments to optimize sampling strategy of resting cysts of Alexandrium catenella to estimate their abundance with a predefined error. This work also aims to provide information on spatial distribution of resting cysts in sediments. The distribution mode of A. catenella resting cysts related to the abundance variability was studied through sediment cores sampling on four different spatial scales and using Ludox CLX gradient density method. The quantification method underestimates by a factor of 2 the resting cysts abundance in one gram of sediment. Application of Taylor's power law allowed us to define a compromise between sampling effort and abundance estimation error. In the case of A. catenella resting cysts from Thau lagoon, the optimal sampling strategy consists of sampling 10 stations on a surface of 2 km² for a given coefficient of variability (C) of 15%, sampling 3 sediment cores at each station (C = 30%) and counting only one replicate by core (C = 18%). Results related to the application of Taylor's power law are closely dependent on resting cyst density and aggregation in a given sediment. In our area, A. catenella resting cysts are mainly observed in the upper 3 cm of sediment. Horizontally, their heterogeneity is lower on 10 cm² surface and tends to stabilize itself beyond a surface of 10 m². Each author has to carry out this pre-sampling effort for his own resting cysts-forming species, in his own area, in order to increase accuracy of resting cyst mapping.     

Development of a quantitative PCR method to explore the historical occurrence of a nuisance microalga under expansion

A number of marine and freshwater harmful algal bloom (HAB) species have colonized new areas and expanded their habitat range in recent years. Nevertheless it is notoriously difficult to establish when colonization first occurred, what the dispersal routes are, and to separate recent invasion from increases in existent but small populations. The freshwater raphidophyte Gonyostomum semen is a nuisance species that has expanded its habitat range and increased in abundance in northern Europe during the past decades. To evaluate to what extent sediments can be used for determining historic occurrence of G. semen, a quantitative real-time PCR method for detecting cysts of this algae was developed. This paper presents a qPCR protocol with a set of primers that are specific to Gonyostomum and with PCR conditions optimized for sediment samples from humic lakes, which are the common habitat of G. semen. With this sensitive method as few as 1.6 cysts per PCR reaction could be reliably quantified, corresponding to 320 cysts per g wet weight sediment. Cysts were present in sediments with ages ranging from years to decades and their persistence allows detection of historic populations up to at least 50 years old. With this qPCR assay it will be possible to trace the presence of G. semen in environments prior to the onset of algae-specific monitoring programs as well as for quantification in water column samples.     

大亚湾海域锥状斯氏藻孢囊形成与萌发的季节变化

锥状斯氏藻(Scrippsiella trochoidea)是南海大亚湾海域优势甲藻。为了解该藻孢囊形成和萌发动态及其对营养细胞种群动态的影响,2001年1月-2002年1月在大亚湾澳头海域用沉积物捕捉器及TFO重力采泥器对其孢囊进行每月一次的周年监测,同时对浮游植物、水温、盐度、溶解氧等也进行了监测。孢囊形成和萌发分别以沉积物捕捉器中的孢囊形成率以及上表层沉积物中空孢囊的百分比来表示。钙质孢囊和非钙质孢囊年平均形成率分别为1.11×104 cysts m-2d-1和2.13×105 cysts m-2d-1。前者在冬季大量形成,而后者在夏季形成较多。孢囊多在春秋季节萌发,夏季萌发较少,而冬季几乎不萌发。在5月份和10月份营养细胞数量峰形成前,孢囊的萌发出现了高峰,而表层沉积物中的孢囊数量及孢囊形成率则在营养细胞数量峰后大幅度上升。由此可见,大亚湾沉积物中该藻孢囊的萌发给水体提供了丰富的营养细胞,反之水体中高密度营养细胞又促使孢囊的大量形成,从而造成了锥状斯氏藻赤潮在大亚湾海域接连发生。     

Improved real-time PCR method for quantification of the abundance of all known ribotypes of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides by comparing 4 different preparation methods

Red tides by the ichthyotoxic dinoflagellate Cochlodinium polykrikoides have caused large scaled mortality of fish and great loss in aquaculture industry in many countries. Detecting and quantifying the abundance of this species are the most critical step in minimizing the loss. The conventional quantitative real-time PCR (qPCR) method has been used for quantifying the abundance of this species. However, when analyzing > 500 samples collected during huge C. polykrikoides red tides in South Sea of Korea in 2014, this conventional method and the previously developed specific primer and probe set for C. polykrikoides did not give reasonable abundances when compared with cell counting data. Thus improved qPCR methods and a new specific primer and probe set reflecting recent discovery of 2 new ribotypes have to be developed. A new species-specific primer and probe set for detecting all 3 ribotypes of C. polykrikoides was developed and provided in this study. Furthermore, because the standard curve between cell abundance and threshold cycle value (Ct) is critical, the efficiencies of 4 different preparation methods used to determine standard curves were comparatively evaluated. The standard curves were determined by using the following 4 different preparations: (1) extraction of DNA from a dense culture of C. polykrikoides followed by serial dilution of the extracted DNA (CDD method), (2) extraction of DNA from each of the serially diluted cultures with different concentrations of C. polykrikoides cultures (CCD method), (3) extraction of DNA from a dense field sample of C. polykrikoides collected from natural seawater and then dilution of the extracted DNA in serial (FDD method), and (4) extraction of DNA from each of the serially diluted field samples having different concentrations of C. polykrikoides (FCD method). These 4 methods yielded different results. The abundances of C. polykrikoides in the samples collected from the coastal waters of South Sea, Korea, in 2014–2015, obtained using the standard curves determined by the CCD and the FCD methods, were the most similar (0.93–1.03 times) and the second closest (1.16–1.33 times) to the actual cell abundances obtained by enumeration of cells. Thus, our results suggest that the CCD method is a more effective tool to quantify the abundance of C. polykrikoides than the conventional method, CDD, and the FDD and FCD methods.     

Encystment of Peridinium gatunense: occurrence, favourable environmental conditions and its role in the dinoflagellate life cycle in a subtropical lake

1. The abundance of cysts of the bloom‐forming dinoflagellate Peridinium gatunense in the sediments of Lake Kinneret and the effects of environmental conditions on encystment were studied in relation to bloom dynamics. Peak cyst formation coincided with the highest growth rate of the population, prior to bloom peak. 2. Peridinium cysts were counted in water and sediment corer samples from 2000 to 2003 and in archived sediment trap samples collected during 1993–94. The cyst data were examined in relation to ambient temperature and nutrient records, and revealed no direct correlation. 3. In laboratory encystment experiments with Peridinium cells collected from the lake, 0.2–3% of the vegetative cells encysted. Temperature, light and cell density had no significant effect on the percentage of encystment. 4. Cysts were always present in the lake sediments but their abundance in ‘non Peridinium’ years was much lower than after a massive bloom. Vegetative cells were always present in the water column after the collapse of the annual dinoflagellate bloom, potentially serving as the inoculum for the next bloom. We propose that the hardy cysts serve as an emergency ‘gene bank’ to initiate population build up following catastrophic die outs.     

Alexandrium pacificum Litaker sp. nov (Group IV): Resting cyst distribution and toxin profile of vegetative cells in Bizerte Lagoon (Tunisia,Southern Mediterranean Sea)

A high spatial resolution sampling of Alexandrium pacificum cysts, along with sediment characteristics (% H₂O, % organic matter (OM), granulometry), vegetative cell abundance and environmental factors were investigated at 123 study stations in Bizerte Lagoon (Tunisia). Morphological examination and ribotyping of cells obtained from a culture called ABZ1 obtained from a cyst isolated in lagoon sediment confirmed that the species was A. pacificum. The toxin profile from the ABZ1 culture harvested during exponential growth phase was simple and composed of the N-sulfocarbamoyl toxins C1 (9.82 pg toxin cell⁻¹), the GTX6 (3.26 pg toxin cell⁻¹) and the carbamoyl toxin Neo-STX (0.38 pg toxin cell⁻¹). The latter represented only 2.8% of the total toxins in this strain.High abundance of A. pacificum cysts correlated with enhanced percentages of water and organic matter in the sediment. In addition, sediment fractions of less than 63 μm were examined as a favorable potential seedbed for initiation of future blooms and outbreaks of A. pacificum in the lagoon. A significant difference in the cyst distribution pattern was recorded among the lagoon's different zones, with the higher cyst abundance occurring in the inner waters. Also, no correlation due to the specific hydrodynamics of the lagoon was observed in the spatial distribution of A. pacificum cysts and vegetative cells.     

Genomic DNA functions as a universal external standard in quantitative real-time PCR

Real-time quantitative PCR (qPCR) is a powerful tool for quantifying specific DNA target sequences. Although determination of relative quantity is widely accepted as a reliable means of measuring differences between samples, there are advantages to being able to determine the absolute copy numbers of a given target. One approach to absolute quantification relies on construction of an accurate standard curve using appropriate external standards of known concentration. We have validated the use of tissue genomic DNA as a universal external standard to facilitate quantification of any target sequence contained in the genome of a given species, addressing several key technical issues regarding its use. This approach was applied to validate mRNA expression of gene candidates identified from microarray data and to determine gene copies in transgenic mice. A simple method that can assist achieving absolute quantification of gene expression would broadly enhance the uses of real-time qPCR and in particular, augment the evaluation of global gene expression studies.     

Development of real-time RT-PCR for detecting viable Cochlodinium polykrikoides (Dinophyceae) cysts in sediment

Morphological observations have confirmed that cysts are produced by dinoflagellates. However, finding a seed bed or unknown cysts in field samples by microscopy is extremely time consuming. Real-time PCR has been used to facilitate the detection of dinoflagellate cysts in sediment. However, DNA from dead vegetative cells remaining on the surface sediment may persist for a long period of time, which can cause false positive DNA detection. In this study, a non-quantitative RNA targeted probe using real-time RT-PCR was developed for detection of viable cysts in sediment. Large-subunit rRNA was used to develop a species-specific RNA targeted probe for the ichthyotoxic dinoflagellate Cochlodinium polykrikoides. The sediment samples were sieved and incubated at 30 °C for 3 h prior to RNA extraction to remove RNA from dead cells remaining in the sediment. Nested-PCR was conducted to maximize assay sensitivity. A field survey to determine the distribution of cysts at 155 sampling stations in the western and southern part of the Korean peninsula showed that C. polykrikoides cysts were detected at five sampling stations.     

Development of a qPCR assay for tracking the ecological niches of genetic sub-populations within Pseudo-nitzschia pungens (Bacillariophyceae)

Three genetic sub-populations (clade I, II and III) of Pseudo-nitzschia pungens, the potential toxic marine diatom, are known to have distinguishable growth characteristics under different culture conditions and distinct distributed patterns in the world. However, to date their exact eco-physiological traits are unrevealed in fields due to lack of the method to detect and/or measure abundances of each sub-populations, hence, the qPCR (quantitative real-time polymerase chain reaction) assay was developed to detect and quantify the P. pungens cells of each clade. Designed two specific primer sets, Pcla12F/R (for clade I and II) and Pcla3F/R (for clade III) only could amplify each target genomic DNA. The, significant linear relationships (R² > 0.998) was established between C_t (threshold cycle) value and the log of cell abundance for each clade. Through the melting curve analysis, comparisons for gene copy numbers among the three clades and spike test for field study, our qPCR assay was reliable to quantify the cell numbers of each clade. There was strong linear correlation (R² > 0.990) between cell abundances as estimated by qPCR assay and direct counting via light microscope in spike test, and 0.24 (clade I), 0.25 (clade II) and 0.33 (clade III) P. pungens cells per mL were detected markedly upon the use of specific two-primer set. Finally, developed qPCR assay was applied on field samples successfully. Our study implicate that our qPCR assay is an accurate and sensitive technique to estimate the cell abundances of each clade of P. pungens in field works.     

Cyst formation,sedimentation, and preservation: Factors affecting dinoflagellate assemblages in recent sediments from trondheimsfjord,Norway

Plankton records and 25 samples of Recent sediment from Trondheimsfjord and the adjoining shelf were studied to investigate production, sedimentation, and preservation of cysts, as factors which influence the eventual composition of dinoflagellate cyst assembleges. All sediment samples were examined for dinoflagellate cysts using routine semiquantitative palynological procedures. In addition, fjord sediments were subjected to a limited sediment analysis, and, for three samples, results from preparations both with and without acid treatments were compared. For the first time, cyst assembleges from Recent sediments were directly compared with extensive plankton records from overlying waters. Results indicate that approximately 20% of the 55 locally recorded dinoflagellate species contribute cysts to bottom sediments. Once formed, cysts behave as fine silt particles in the sedimentary regime, increasing in abundance as the percentage abundance of finer sediment increases, usually with increased water depth. Cyst-forming species are almost entirely restricted to a few genera, particularly Gonyaulax and Peridinium, within the order Peridiniales. For some groups, reasonably good correspondence was found between percentage abundances of dinoflagellates in plankton and their cysts in sediment, though plankton records covering at least five years were required to establish this. Gonyaulax grindleyi Reinecke (Von Stosch 1969) appeared to be consistently overrepresented by cysts in sediment relative to available plankton evidence; possible explanations are suggested. At least 30% of the cyst species present, including most Peridinium species, were eliminated, or rendered unreliable for semiquantitative palynology, by application of routine palynological preparation treatments. Such cysts may provide useful, non-quantitative, palynological information from Recent and possibly Quaternary sediments, but their persistence would seem unlikely. Thus, factors of preservation probably further restrict the dinoflagellate fossil record. Cyst assemblages from Trondheimsfjord are comparable with those previously recorded from the northeastern coast of U.S.A., and from Scotland and northeastern England. Fjord assemblages are dominated by small, simple, spinose cysts which would be regarded as acritarchs if culture experiments had not proved that they are dinoflagellate cysts. Much potential biogeographic and palaeoenvironmental information was included within the less abundant species.Attention is drawn to the role which future culture experiments may be expected to play in helping to resolve taxonomic difficulties currently affecting dinoflagellate studies. Palynological significance of results from the present study is discussed especially with reference to recent work by Von Stosch which strongly suggests that cysts may be hypnozygotes formed routinely in sexual cycles of dinoflagellates.     

Real-Time PCR for Quantification of Giardia and Cryptosporidium in Environmental Water Samples and Sewage

The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the β-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.     

Sinking of Heterosigma akashiwo results in increased toxicity of this harmful algal bloom species

Notable physiological responses such as toxicity and sinking rates of the red tide forming raphidophyte Heterosigma akashiwo are correlated with high levels of macronutrient stress. Individual cells of this species are also capable of forming benthic vegetative cysts that overwinter in marine sediment and contribute to bloom propagation in subsequent seasons. It was hypothesized that there is variability in the rates of sinking within cell cultures and that sinking cells are more toxic than the neutrally buoyant or floating cells. Using laboratory-based settling columns, various isolates of H. akashiwo were allowed to separate, and the toxicities of sinking and floating populations were analyzed. Sinking and floating rates were significantly higher during the late stationary growth phase for all isolates. For two H. akashiwo isolates, sinking populations were significantly more toxic than those that were positively buoyant. A similar trend was observed in a third strain, however the relationship was not significant. Differences in adaptive ecophysiology among the different strain likely caused the variation. It is suggested that the most toxic cells within a bloom are those found at the lower depths, potentially interacting with the benthic community or ensuring that subsequent bloom propagation contains cells with the potential for toxicity.     

Tuber aestivum Vittad. mycelium quantified: advantages and limitations of a qPCR approach

A quantitative real-time PCR (qPCR) marker Ta0 with hydrolysis probe (“TaqMan”), targeted to the internal transcribed spacer region of the ribosomal DNA, has been developed for quantification of summer truffle (Tuber aestivum) mycelium. Gene copy concentrations determined by the qPCR were calibrated against pure culture mycelium of T. aestivum, enabling quantification of the mycelium in soil and in host roots from the fields. Significant concentrations of the fungus were observed not only in the finest roots with ectomycorrhizae but also in other root types, indicating that the fungus is an important component of the microbial film at the root surface. The concentration of T. aestivum in soil is relatively high compared to other ectomycorrhizal fungi. To evaluate the reliability of the measurement of the soil mycelium density using qPCR, the steady basal extracellular concentration of the stabilized T. aestivum DNA should be known and taken into account. Therefore, we addressed the stability of the qPCR signal in soil subjected to different treatments. After the field soil was sieved, regardless of whether it was dried/rewetted or not, the T. aestivum DNA was quickly decomposed. It took just about 4 days to reach a steady concentration. This represents a conserved pool of T. aestivum DNA and determines detection limit of the qPCR quantification in our case. When the soil was autoclaved and recolonized by saprotrophic microorganisms, this conserved DNA pool was eliminated and the soil became free of T. aestivum DNA.     

Towards an optimal sampling strategy for Alexandrium catenella (Dinophyceae) benthic resting cysts

The study proposes methodological developments to optimize sampling strategy of resting cysts of Alexandrium catenella to estimate their abundance with a predefined error. This work also aims to provide information on spatial distribution of resting cysts in sediments. The distribution mode of A. catenella resting cysts related to the abundance variability was studied through sediment cores sampling on four different spatial scales and using Ludox CLX gradient density method. The quantification method underestimates by a factor of 2 the resting cysts abundance in one gram of sediment. Application of Taylor's power law allowed us to define a compromise between sampling effort and abundance estimation error. In the case of A. catenella resting cysts from Thau lagoon, the optimal sampling strategy consists of sampling 10 stations on a surface of 2 km² for a given coefficient of variability (C) of 15%, sampling 3 sediment cores at each station (C = 30%) and counting only one replicate by core (C = 18%). Results related to the application of Taylor's power law are closely dependent on resting cyst density and aggregation in a given sediment. In our area, A. catenella resting cysts are mainly observed in the upper 3 cm of sediment. Horizontally, their heterogeneity is lower on 10 cm² surface and tends to stabilize itself beyond a surface of 10 m². Each author has to carry out this pre-sampling effort for his own resting cysts-forming species, in his own area, in order to increase accuracy of resting cyst mapping.     

Application of real-time PCR assay for detection and quantification of Alexandrium tamarense and Alexandrium catenella cysts from marine sediments

The dinoflagellates Alexandrium tamarense (Lebor) Balech and Alexandrium catenella (Whedon and Kofoid) Balech (Dinophyceae) are believed to be the main species responsible for paralytic shellfish poisoning (PSP) all over the world. It is necessary to identify A. tamarense and A. catenella cysts and to monitor their distribution in sediment in order to minimize the damages caused by PSP to the economy and food quality because cysts are the seed population for blooms caused by motile vegetative cells. In this study, we developed an efficient DNA extraction method from the natural cysts present in marine sediments after they were size fractionated with a plankton net (mesh size of 20–150 μm). The 10–3000 cysts were added to the sediments collected from the Ariake Sea, and for which the primuline-staining method did not reveal any cysts. DNA was then extracted from each sample, and linear standard curves for A. tamarense and A. catenella cysts were obtained from the correlation between the Ct values by real-time PCR and the log of the initial densities of cysts. We monitored the A. tamarense and A. catenella cyst densities in the environmental samples. This assay was demonstrated to be a powerful tool for the identification, detection, and quantification of the cysts of the toxic dinoflagellates.     

Comparison between Culture and a Multiplex Quantitative Real-Time Polymerase Chain Reaction Assay Detecting Ureaplasma urealyticum and U. parvum

A novel multiplex quantitative real-time polymerase chain reaction (qPCR) for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard''s 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR.     

Enhancing the Detection of Giardia duodenalis Cysts in Foods by Inertial Microfluidic Separation

The sensitivity and specificity of current Giardia cyst detection methods for foods are largely determined by the effectiveness of the elution, separation, and concentration methods used. The aim of these methods is to produce a final suspension with an adequate concentration of Giardia cysts for detection and a low concentration of interfering food debris. In the present study, a microfluidic device, which makes use of inertial separation, was designed and fabricated for the separation of Giardia cysts. A cyclical pumping platform and protocol was developed to concentrate 10-ml suspensions down to less than 1 ml. Tests involving Giardia duodenalis cysts and 1.90-μm microbeads in pure suspensions demonstrated the specificity of the microfluidic chip for cysts over smaller nonspecific particles. As the suspension cycled through the chip, a large number of beads were removed (70%) and the majority of the cysts were concentrated (82%). Subsequently, the microfluidic inertial separation chip was integrated into a method for the detection of G. duodenalis cysts from lettuce samples. The method greatly reduced the concentration of background debris in the final suspensions (10-fold reduction) in comparison to that obtained by a conventional method. The method also recovered an average of 68.4% of cysts from 25-g lettuce samples and had a limit of detection (LOD) of 38 cysts. While the recovery of cysts by inertial separation was slightly lower, and the LOD slightly higher, than with the conventional method, the sample analysis time was greatly reduced, as there were far fewer background food particles interfering with the detection of cysts by immunofluorescence microscopy.     

Distributions of calcareous dinoflagellate cysts in surface sediments of the western equatorial Atlantic Ocean,and their potential use in palaeoceanography

Only very few studies focus on recent calcareous dinoflagellate cyst diversity, geographic distribution and ecology, so that information on the distribution patterns and environmental affinities of individual cyst species is extremely limited. This information is, however, essential if we want to use calcareous dinoflagellate cysts for palaeoenvironmental reconstruction. Surface sediment samples from the generally oligotrophic western equatorial Atlantic Ocean, offshore northeast Brazil, were therefore quantitatively analysed for their calcareous dinoflagellate cyst content, including the calcareous vegetative coccoid Thoracosphaera heimii. Seven calcareous dinoflagellate cyst species/morphotypes and T. heimii were encountered in high concentrations throughout the area. Substantial differences in the distribution patterns were observed. The highest concentrations of cysts are found in sediments of the more oligotrophic, oceanic regions, beyond the influence of Amazon River discharge waters. Dinoflagellates producing calcareous cysts thus appear to be capable of surviving low nutrient concentrations and produce large numbers of cysts in relatively stable and predictable environments affected by minimal seasonality. To test for the environmental affinities of individual species, distribution patterns in surface sediments were compared with temperature, salinity, density and stratification gradients within the upper water column (0–100 m) over different times of the year, using principal components analysis and redundancy analysis. T. heimii and four of the seven encountered cyst species (Sphaerodinella? albatrosiana, two morphotypes of Sphaerodinella? tuberosa and Scrippsiella regalis) relate to these parameters significantly and the variations in the cyst associations appear to be associated with the different surface water currents characterising the area. The results imply that calcareous dinoflagellate cyst distributions can potentially be used to distinguish between different open oceanic environments and they could, therefore, be useful in tracing water mass movements throughout the late Quaternary.     

Control of modern dinoflagellate cyst distribution in the Irish and Celtic seas by seasonal stratification dynamics

Surface sediments from seven stations located in the seasonally stratified, frontal and mixed water regions in the Celtic and Irish seas have been analysed for their dinoflagellate cyst assemblages and dinosterol content. A total of 45 dinoflagellate cyst taxa have been identified and the assemblages related to surface and sediment conditions. Sediments from the mixed water region, at 30 m water depth, are characterised by a relatively low cyst concentration (∼2300 cysts/g dry weight) and high relative abundances of Lingulodinium machaerophorum accompanied by Spiniferites membranaceus, Brigantedinium spp. and Dubridinium caperatum. Assemblages from stratified and frontal water stations are dominated by Spiniferites ramosus associated with Operculodinium centrocarpum, Brigantedinium spp., cysts of Polykrikos schwartzii and Selenopemphix quanta. Ordination techniques performed on a restricted number of 35 taxa from the assemblages differentiated the stratified and frontal assemblages based on the abundance of the less abundant species Bitectatodinium tepikiense and Spiniferites elongatus. Among the environmental parameters (sea-surface temperature and salinity, stratification index, chlorophyll concentration and sediment grain-size classes), the seasonal stratification and sedimentological context, itself a function of tidal dynamics, explain most of the variance in the environmental conditions. These results indicate that dinoflagellate cyst analyses of shelf sediment records can be used to document the planktonic signal of seasonal stratification dynamics.     

Mapping the Distribution of Cysts from the Toxic Dinoflagellate Cochlodinium polykrikoides in Bloom-Prone Estuaries by a Novel Fluorescence In Situ Hybridization Assay

Cochlodinium polykrikoides is a cosmopolitan dinoflagellate that is notorious for causing fish-killing harmful algal blooms (HABs) across North America and Asia. While recent laboratory and ecosystem studies have definitively demonstrated that Cochlodinium forms resting cysts that may play a key role in the dynamics of its HABs, uncertainties regarding cyst morphology and detection have prohibited even a rudimentary understanding of the distribution of C. polykrikoides cysts in coastal ecosystems. Here, we report on the development of a fluorescence in situ hybridization (FISH) assay using oligonucleotide probes specific for the large subunit (LSU) ribosomal DNA (rDNA) of C. polykrikoides. The LSU rDNA-targeted FISH assay was used with epifluorescence microscopy and was iteratively refined to maximize the fluorescent reaction with C. polykrikoides and minimize cross-reactivity. The final LSU rDNA-targeted FISH assay was found to quantitatively recover cysts made by North American isolates of C. polykrikoides but not cysts formed by other common cyst-forming dinoflagellates. The method was then applied to identify and map C. polykrikoides cysts across bloom-prone estuaries. Annual cyst and vegetative cell surveys revealed that elevated densities of C. polykrikoides cysts (>100 cm⁻³) during the spring of a given year were spatially consistent with regions of dense blooms the prior summer. The identity of cysts in sediments was confirmed via independent amplification of C. polykrikoides rDNA. This study mapped C. polykrikoides cysts in a natural marine setting and indicates that the excystment of cysts formed by this harmful alga may play a key role in the development of HABs of this species.