Rapid quantification of the toxic alga Prymnesium parvum in natural samples by use of a specific monoclonal antibody and solid-phase cytometry

The increasing incidence of harmful algal blooms around the world and their associated health and economic effects require the development of methods to rapidly and accurately detect and enumerate the target species. Here we describe use of a solid-phase cytometer to detect and enumerate the toxic alga Prymnesium parvum in natural samples, using a specific monoclonal antibody and indirect immunofluorescence. The immunoglobulin G antibody 16E4 exhibited narrow specificity in that it recognized several P. parvum strains and a Prymnesium nemamethecum strain but it did not cross-react with P. parvum strains from Scandinavia or any other algal strains, including species of the closely related genus Chrysochromulina. Prymnesium sp. cells labeled with 16E4 were readily detected by the solid-phase cytometer because of the large fluorescence signal and the signal/noise ratio. Immunofluorescence detection and enumeration of cultured P. parvum cells preserved with different fixatives showed that the highest cell counts were obtained when cells were fixed with either glutaraldehyde or formaldehyde plus the cell protectant Pluronic F-68, whereas the use of formaldehyde alone resulted in significantly lower counts. Immunofluorescence labeling and analysis with the solid-phase cytometer of fixed natural samples from a bloom of P. parvum occurring in Lake Colorado in Texas gave cell counts that were close to those obtained by the traditional method of counting using light microscopy. These results show that a solid-phase cytometer can be used to rapidly enumerate natural P. parvum cells and that it could be used to detect other toxic algae, with an appropriate antibody or DNA probe.     

Automated detection and enumeration for toxic algae by solid-phase cytometry and the introduction of a new probe for Prymnesium parvum (Haptophyta: Prymnesiophyceae)

Harmful algal blooms have a severe impact on aquaculture andfishery and can be caused by toxic haptophytes and dinoflagellates.Different toxic species, which are not easy to distinguish fromtheir morphologically similar and non-toxic relatives, occurin both groups. Sequencing of the large subunit ribosomal RNAof different strains and taxonomic relatives allowed the designof a probe specific to the toxic Prymnesium parvum spp. Forthe rapid detection and enumeration of Prymnesium and Alexandriumcells in cultures and environmental samples, respectively, protocolsfor fluorescence in situ hybridization were adapted for automateddetection by a solid-phase cytometer, the ChemScan. This cytometerenables the automated counting of fluorescently labelled cellson a membrane filter and subsequently a microscopic verificationof these results by the user, because the motorized stage ofthe microscope is driven to each positive signal by the computersoftware to localize that cell on the filter. With this fastdetection method, it was possible to detect, enumerate and verifymicroalgal cells on a filter, with a detection limit of onecell per membrane filter.     

A-, B- and C-type prymnesins are clade specific compounds and chemotaxonomic markers in Prymnesium parvum

Harmful blooms formed by planktonic microalgae (HABs) in both freshwater and coastal waters regularly lead to severe mortalities of fish and invertebrates causing substantial economic losses of marine products worldwide. The mixotrophic haptophyte Prymnesium parvum is one of the most important microalgae associated with fish kills. Here 26 strains of P. parvum with a wide geographical distribution were screened for the production of prymnesins, the suspected causative allelochemical toxins. All investigated strains produced prymnesins, indicating that the toxins play an important role for the organism. The prymnesins can be classified into three types based on the length of the carbon backbone of the compound and each algal strain produced only one of these types. Biogeographical mapping of the prymnesin distribution indicated a global distribution of each type. In addition, phylogenetic analyses based on internal transcribed spacer (ITS) sequences revealed monophyletic origin of all prymnesin types and clades could therefore be defined based on the toxic compound. It might be that evolution of new species within the P. parvum species complex is driven by changes in toxin type or that they are a result of it. Such a correlation between chemotype and phylotype has never been documented before for a harmful microalga. Chemotaxonomy and ITS-type classification may thus be used to further delimit the P. parvum species complex.     

Identification of toxic fatty acid amides isolated from the harmful alga Prymnesium parvum carter

The golden alga Prymnesium parvum has been implicated in fish and aquatic animal kills globally for over a century. In addition to widespread ecological impacts through the loss of entire fish populations within lakes, an economic burden is also felt by state and local agencies due to year class losses of fish raised for stocking lakes as well as loss of fishing and recreational use of the affected water. Multiple compounds have been implicated in P. parvum toxicity, but the unequivocal identification and characterization of all P. parvum toxins remained to be accomplished. To unambiguously characterize these toxins, we analyzed laboratory-cultured cells exposed to limited nitrogen and phosphorus concentrations, uni-algal wild cells collected from an ichthytoxic bloom event at Lake Wichita, TX, and the water from both cultured and field-collected algae. A bioassay-guided fractionation process was employed to chemically isolate P. parvum toxins using both mammalian cells and larval fish. The results of these assays revealed that there was a distinct similarity in the toxic compounds characterized as seven primary fatty acid amides (myristamide, palmitamide, linoleamide, oleamide, elaidamide, stearamide, and erucamide) and one hydroxamic acid (linoleyl hydroxamic acid). These compounds display cytotoxic and ichthytoxic activity and have not yet been reported in P. parvum toxicity or in the toxicity of harmful algal species.     

The effects of aeration on growth and toxicity of Prymnesium parvum grown with and without algal prey

We investigated the effects of aeration on growth and toxicity of the haptophyte Prymnesium parvum in the presence and absence of the algal prey Rhodomonas salina. Batch monocultures of P-limited P. parvum and N and P sufficient R. salina and mixed cultures of the two microalgae were grown with no, low (20) and high (100) ml min⁻¹ aeration for 18 days. Cell growth of P. parvum and R. salina and cell toxicity of P. parvum were studied over the experimental period. The highest specific growth rates of P. parvum were found at low aeration rates. R. salina in monocultures showed typical growth patterns, while R. salina numbers declined rapidly in the mixed cultures. Of the initial cell densities, 98–100% of the R. salina cells were lysed or ingested within 24 h of mixing with P. parvum cells. The maxima P. parvum biomasses were significantly higher in the mixed cultures than in the monocultures. Cell toxicity of P. parvum increased significantly in response to aeration rates and the highest levels were found in the high aeration condition. Availability of prey and resupply of inorganic nutrients decreased P. parvum cell toxicity. Our study suggests that P. parvum is tolerant and is able to grow over a broad range of aeration and associated turbulence effects though low aeration represents an optimal condition for growth. As P. parvum toxicity was higher in the high aeration treatment we suggest that the higher concentrations of oxygen cause more toxins to be produced, as these are oxygen rich compounds. We suggest that oxygenation and turbulence of surface waters caused by mixing may be involved in promoting high toxic P. parvum blooms in shallow lakes and coastal waters.     

Beyond hydraulic flushing: Deep water mixing takes the harm out of a haptophyte algal bloom

Inflows are linked to water quality, food web dynamics, and the incidence of harmful algal blooms (HABs). It may be that inflows can be manipulated to mitigate some blooms and accelerate recovery of living natural resources, such as fisheries. Lack of available water, however, can limit this approach to management. Utilizing source water from deeper depths to displace surface waters, however, might effectively mimic aspects of inflow events, such as disrupting ecological processes and community succession through hydraulic displacement. We tested this notion by conducting in-lake mesocosm experiments with natural plankton communities plagued by Prymnesium parvum where we manipulated hydraulic flushing. We found that P. parvum cell densities were reduced by up to 69% and 53%, and ambient toxicity ameliorated, during pre-bloom and bloom development periods. Furthermore, other phytoplankton taxa and zooplankton benefited from these pulsed flushing events. In other words, hydraulic flushing with deep waters not only suppressed P. parvum bloom initiation and development, but also proved beneficial to other aspects of the lower food web. These observations provide the first demonstration that P. parvum bloom initiation may be interrupted, and bloom development may be arrested, through hydraulic manipulations.     

Multiplex PCR methods for the species-specific detection and quantification of <Emphasis Type="Italic">Prymnesium parvum</Emphasis> (Haptophyta)

Multiplex polymerase chain reaction (PCR) assays were developed for detecting and quantifying Prymnesium parvum wherein suites of primers simultaneously amplify four species- and gene-specific products using genomic DNA or whole cells for template. With conventional PCR, amplification products were easily resolved by gel electrophoresis, generating a diagnostic banding pattern. Gene-specific fluorescent molecular beacons were designed for use with real-time quantitative PCR (qPCR). Both methods were capable of detecting as few as one or two cells in 50 cycles. The species and gene specificities of the assays were evaluated using isolates (and mixtures) of P. parvum, related species, and out-groups. Cell counts using qPCR to evaluate environmental samples were comparable to mean values obtained from manual counts and had lower standard deviations. This presents a significant improvement in DNA-based detection technology, enhanced by the rapid and simultaneous confirmation of four species-specific products and the ability to detect several widely separated geographic isolates of P. parvum.     

Behavioural differences underlie toxicity and predation variation in blooms of Prymnesium parvum

Much of the evolutionary ecology of toxic algal blooms (TABs) remains unclear, including the role of algal toxins in the adaptive ‘strategies’ of TAB-forming species. Most eukaryotic TABs are caused by mixotrophs that augment autotrophy with organic nutrient sources, including competing algae (intraguild predation). We leverage the standing diversity of TABs formed by the toxic, invasive mixotroph Prymnesium parvum to identify cell-level behaviours involved in toxin-assisted predation using direct observations as well as comparisons between genetically distinct low- and high-toxicity isolates. Our results suggest that P. parvum toxins are primarily delivered at close range and promote subsequent prey capture/consumption. Surprisingly, we find opposite chemotactic preferences for organic (prey-derived) and inorganic nutrients between differentially toxic isolates, respectively, suggesting behavioural integration of toxicity and phagotrophy. Variation in toxicity may, therefore, reflect broader phenotypic integration of key traits that ultimately contribute to the remarkable flexibility, diversity, and success of invasive populations.     

The hidden flora of a lake

Summary Twenty-seven water samples taken from the Sea of Galilee, Israel, were enriched selectively with an inorganic salt medium to further the appearance of halophilic algae, rare or unknown from the lake. The Chrysophycean flagellatePrymnesium parvum, known to cause toxic blooms, has been especially looked for.A radical change in the algal composition of the enriched samples could be observed. Out of 31 species that proliferated in the so established cultures, 11 were new to the lake. Fifteen species belonged to the Cyanophyceae, 8 to the Diatomeae, and 7 species belonged to the Chlorophyceae. Prymnesium parvum could be found in 6 out of the 27 samples.It is concluded that in case of a sufficient rise in the lake's salinity, halophilic algae that are now rare or dormant in the lake, will replace the present algal flora. The presence in the lake ofPrymnesium parvum forms a potential danger of toxic blooms.

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Résumé Vingt-sept échantillons d'eau prélevés dans le Lac de Tibériade, Israel, ont été enrichis sélectivement par des milieux salins inorganiques afin de stimuler le développement d'algues halophiles rares ou inconnues dans le lac. Notre attention a été particulièrement attirée par la présence du Chrysophyte flagélléPrymnesium parvum, qui provoque des pullulations toxiques dans L'eau.Nous avons pu observer un changement radical dans la composition floristique des échantillons enrichis. Parmi les 31 espèces qui se sont développées dans nos cultures, 11 espèces sont inconnues dans le lac; 15 espèces appartiennent aux Cyanophyceae, 8 aux Diatomées et 7 aux Chlorophyceae. Nous avons trouvéPrymnesium parvum dans 6 échantillons sur 27.Nous pouvons conclure, qu'en cas d'accroissement du taux de la salinité dans le lac, les algues halophiles, actuellement rares ou dormantes, remplaceront la flore présente.Les possibles pullulations toxiques dePrymnesium parvum dans les eaux du Lac constituent donc un danger permanent.

     

Incorporating molecular tools into routine HAB monitoring programs: Using qPCR to track invasive Prymnesium

Microscopy, a staple of monitoring programs for tracking the occurrence and abundance of harmful algal bloom (HAB) species, is time consuming and often characterized by high uncertainty. Alternate methods that allow rapid and accurate assessment of presence and abundance of HAB species are needed. For many HAB species, such as the toxigenic haptophyte, Prymnesium parvum, molecular methods including quantitative real-time PCR (qPCR) have been developed with the suggestion that they should be useful for monitoring programs. However, this suggestion rarely has been put into action. In this study, we modified a recently developed method for detecting P. parvum using qPCR and tested its efficacy as an alternative to microscopy for P. parvum detection and enumeration in a long-term monitoring program in a recently invaded subtropical US reservoir. Abundance estimates of P. parvum were similar for both methods, but we detected P. parvum at multiple sites using qPCR where it previously had gone undetected by microscopy. Using qPCR, we substantially reduced processing time, increased detection limit and reduced error in P. parvum abundance estimates compared to microscopy. Thus, qPCR is an effective tool for detecting and monitoring P. parvum, particularly at pre-bloom densities, and should likewise prove useful in monitoring programs for the other HAB species for which qPCR methods have been developed.     

Feeding by ciliates on two harmful algal bloom species, Prymnesium parvum and Prorocentrum minimum

We have focused on ciliates as potential grazers on toxic phytoplankton because they are major herbivores in aquatic food webs. Ciliates may exert top down control on toxic phytoplankton blooms, potentially suppressing or shortening the duration of harmful algal blooms (HABs). We measured the growth rates of several ciliate species on uni-algal and mixed diets of both HAB and non-HAB algae. The tintinnids Favella ehrenbergii, Eutintinnus pectinis and Metacylis angulata and the non-loricate ciliates Strombidinopsis sp. and Strombidium conicum were isolated from Long Island Sound (LIS), and fed HAB species including the prymnesiophyte Prymnesium parvum (strain 97-20-01) and the dinoflagellate Prorocentrum minimum (strains Exuv and JA 98-01). Ciliates were fed algal prey from cultures at various growth phases and at varying concentrations. We observed no harmful effects of P. minimum (Exuv) on any of the ciliates. However in a comparison of strains, P. minimum (Exuv) supported high growth rates, whereas P. minimum (JA 98-01) supported only nominal growth. P. parvum was acutely toxic to ciliates at high concentrations (2×10⁴–3×10⁴ cells ml⁻¹). At low concentrations (5×10³–1×10⁴ cells ml⁻¹), or in culture filtrate, ciliates survived for at least several hours. In mixed diet experiments, as long as a non-toxic alga was available, ciliates survived and at times grew well at concentrations of P. parvum (5×10²–3×10⁴ cells ml⁻¹) that would otherwise have killed them. The present study suggests that prior to the onset of toxicity and bloom formation ciliates may exert grazing pressure on these HAB species, potentially contributing to the suppression or decline of P. minimum and P. parvum blooms.     

Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR

Aims: The ichthyotoxic species Prymnesium parvum (Haptophyceae) is difficult to quantify in a microscopy‐based monitoring programme, because the cells are very small, fragile and their morphology can be distorted by the use of fixatives. In the attempt to overcome these problems, a real‐time PCR‐based method for the rapid and sensitive identification and quantification of P. parvum was developed. Methods and Results: A quantitative real‐time PCR assay was optimized with primers designed on the internal transcribed spacer 2 rDNA region of P. parvum. This PCR assay was specific, showing no amplification of DNA extracted from closely related species, and sensitive. Moreover, this method was able to detect and reliably quantify P. parvum cells in preserved environmental samples artificially spiked with known amounts of cultured cells. Conclusions: Considering the specificity, sensitivity and applicability to preserved environmental samples, this method may be a useful tool for the monitoring of this toxic species. Significance and Impact of the Study: The real‐time PCR method described in this study may represent a progress towards the rapid detection and quantification of P. parvum cells in water‐monitoring programmes, allowing the early application of strategies to control bloom events, such as the use of clay minerals.     

Stability of the intra- and extracellular toxins of Prymnesium parvum using a microalgal bioassay

Prymnesium parvum produces a variety of toxic compounds, which affect other algae, grazers and organisms at higher trophic levels. Here we provide the method for development of a sensitive algal bioassay using a microalgal target, Teleaulax acuta, to measure strain variability in P. parvum toxicity, as well as the temporal stability of both the intracellular and the extracellular lytic compounds of P. parvum. We show high strain variation in toxicities after 3 h incubation with LC₅₀s ranging from 24 to 223 × 10³ cells ml⁻¹. Most importantly we prove the necessity of testing physico-chemical properties of P. parvum toxins before attempting to isolate and characterize them. The extracellular toxin in the supernatant is highly unstable, and it loses significant lytic effects after 3 days despite storage at −20 °C and after only 24 h stored at 4 °C. However, when stored at −80 °C, lytic activity is more easily maintained. Reducing oxidation by storing the supernatant with no headspace in the vials significantly slowed loss of activity when stored at 4 °C. We show that the lytic activity of the intracellular toxins, when released by sonication, is not as high as the extracellular toxins, however the stability of the intracellular toxins when kept as a cell pellet at −20 °C is excellent, which proves this is a sufficient storage method for less than 3 months. Our results provide an ecologically appropriate algal bioassay to quantify lytic activity of P. parvum toxins and we have advanced our knowledge of how to handle and store the toxins from P. parvum so as to maintain biologically relevant toxicity.     

Prymnesium parvum invasion success into coastal bays of the Gulf of Mexico: Galveston Bay case study

The toxic haptophyte Prymnesium parvum regularly forms fish-killing blooms in inland brackish water bodies in the south-central USA. Along the Texas coast smaller blooms have occurred in isolated areas. There appears to be an increasing risk that harmful P. parvum blooms will propagate into open coastal waters with implementation of future water plans. These plans will include increased interbasin water transfers from the Brazos River, regularly impacted by P. parvum blooms, to the San Jacinto-Brazos Coastal Basin, which ultimately flows into Galveston Bay (GB). Persisting source populations of P. parvum in inland waters elevates this risk. Thus, there is a need for an increased understanding of how P. parvum might perform in coastal waters, such as those found in GB. Here, two in-field experiments were conducted to investigate the influence of various plankton size-fractions of GB water on inoculated P. parvum during fall and winter, periods when blooms are typically initiating and developing inland. Stationary- and log-growth phase P. parvum were used to represent high and low toxicity initial conditions. Results revealed that P. parvum could grow in GB waters and cause acute mortality to silverside minnows (Menidia beryllina). Depending on season and growth phase, however, P. parvum growth and toxicity varied in different size fractions. During the fall, P. parvum inoculated from stationary-, but not log-growth phase culture, was negatively affected by bacteria-sized particles. During the winter, bacteria and nanoplankton together had a negative effect on P. parvum inoculated from stationary- and, to a lesser degree, log-growth phase cultures. Intermediate- and large-sized grazers when combined with bacteria and nanoplankton had complex relationships with inoculated P. parvum, sometimes stimulating and sometimes suppressing population growth. Toxicity to fish occurred in almost all plankton size fractions. The inclusion of progressively larger sized plankton fractions resulted in trends of decreased toxicity in treatments inoculated with stationary-, but not log-growth phase P. parvum in the fall. In the winter, however, inclusion of larger sized plankton fractions resulted in trends of increased toxicity to fish in treatments inoculated with both stationary- and log-growth phase P. parvum. This study indicates that understanding P. parvum population dynamics in open waters of estuaries and bays will be challenging, as there appears to be complex relationships with naturally occurring components of the plankton. The observations that P. parvum is able to grow to high population density and produce fish-killing levels of toxins underscores the need for advanced risk assessment studies, especially in light of water use plans that will result in P. parvum invasions of greater size.     

Occurrence of toxic Prymnesium parvum blooms with high protease activity is related to fish mortality in Hungarian ponds

The unicellular alga Prymnesium parvum has been responsible for toxic incidents with severe ecological impacts in many parts of the world, and causes massive fish kills worldwide. Recently the haptophyte microalgae have caused water-bloom (4.3 × 10⁴ cells ml⁻¹) in 6 fish ponds with high conductivity in Hungary, and caused fish mortality with typical symptoms. Toxicity of P. parvum from water samples was quantified by the assay of the influence of its cell-free filtrates on haemolysis (346 ± 42.2) and in fish and daphnia toxicity tests. High amount of proteases in P. parvum containing waterbloom samples were detected with the help of activity gel electrophoresis. The proteases of investigated P. parvum samples (125–18 kDa) showed high gelatinolytic activity and some of them showed sensitivity to EDTA (inhibitors of metalloproteases) and to PMSF (inhibitors of serine proteases).     

Removal of Prymnesium parvum (Haptophyceae) cells under different nutrient conditions by clay

In this study, we have shown that the ichthyotoxic Prymnesium parvum (haptophyte) can be successfully removed by spraying the surface with a phosphatic clay/polyaluminium chloride (PAC) mix (final concentration, 4 g l⁻¹ and 5 ppm, respectively). The alga was grown in non-axenic batch cultures with nitrogen deficient, phosphorus deficient and nutrient sufficient media. Sub-samples of the nutrient sufficient culture were diluted to obtain cell abundances equal to those in the nutrient deficient cultures. Clay/PAC removed up to 100% of the cells in the low cell nutrient sufficient treatment after 72 h, but removal in the other treatments was lower (up to 84%). The nitrogen deficient cells were found to be the most toxic, measured as haemolytic activity of the cells (HE₅₀), just prior to the start of the experiment. However, the toxicity of the cells in all treatments was found to fluctuate during the incubation time with a general increase in toxicity towards the end, suggesting that the cells became stressed during sedimentation and/or when trapped in the clay. But the amount of released toxins was always below the detection limit of the haemolytic assay, and the abundance of free-living bacteria, derived from flow cytometer counts, increased throughout the experiment. This suggests that released toxins were either trapped by the clay particles or effectively degraded by the bacterial community. The study shows that the clay method can be efficient in mitigation of P. parvum blooms, but further studies have to be conducted where optimum clay concentrations are determined to ensure that the efficiency is high against nutrient deficient cells and at high cell abundances, i.e. conditions found during blooms in eutrophic coastal waters, and to determine the fate of the cells and their toxins during and after sedimentation.     

Increase in the production of allelopathic substances by Prymnesium parvum cells grown under N- or P-deficient conditions

The haptophyte Prymnesium parvum is known to produce a set of highly potent exotoxins, commonly called prymnesins. These toxins have been shown to have several biological activities, including ichthyotoxic, neurotoxic, cytotoxic, hepatotoxic and hemolytic activity towards a range of marine organisms. In addition, recent studies have shown that the toxicity of P. parvum is enhanced when the cells are grown under N- or P-deficient conditions. In this study, the influence of prymnesium toxins on the growth of other phytoplankton species was investigated by addition of cell-free filtrate of P. parvum cultures grown under nutrient-deficient (N or P) or non-deficient conditions. Addition of cell-free filtrate from P. parvum cultures grown under N or P limitation inhibited the growth of Thalassiosira weissflogii, Prorocentrum minimum and Rhodomonas cf. baltica. In contrast, a strain of Prymnesium patelliferum known to produce prymnesium toxins was not negatively affected under any conditions. Furthermore, addition of filtrates from nutrient-sufficient P. parvum cultures did not negatively influence the growth of any of the tested species. These findings suggest that prymnesium toxins may play an allelopathic role, and that the production of allelopathic substances is regulated by the availability of nutrients.     

INTER- AND INTRASPECIFIC GENETIC VARIATION IN TWELVE PRYMAESIUM (HAPTOPHYCEAE) CLONES1

The haptophytes Prymnesium parvum Carter and Prymnesium patelliferum Green, Hibberd, and Pienaar are two closely related species, which can only be distinguished by minor differences in the morphology of their organic body scales. The two Prymnesium species are reported to coexist at several locations, including the Sands-fjord system in southwestern Norway. Comparisons of physiology and toxicity within the two species have failed to reveal differences that can add to the small morphological distinctions used to separate them. To investigate the genetic relationship between the two species, we compared the sequence of the first internal transcribed spacer region (ITS1)and length variation in one intron separating calmodulin genes for four P. parvum strains and eight P. patelliferum strains. Both the ITS1 sequence and the banding patterns obtained by PCR amplification of one intron in the calmodulin genes indicated that the Prymnesium isolates are related by their geographic origin instead of 4 their species affiliation. The results indicate that P. parvum and P. patelliferum are so closely related that they could be considered one species. Alternatively, we discuss the possibility that the two species might be joined in a heteromorphic haploid-diploid life cycle, as is now widely reported for other haptophycean algae.     

Examination of six commonly used laboratory fixatives in HAB monitoring programs for their use in quantitative PCR based on Taqman probe technology

Due to the need for more rapid and reliable detection, quantification and enumeration of harmful algal species the use of molecular methods are increasingly being used in monitoring and field studies. However, many studies often require sample fixation to allow for transportation before analyses are conducted. Here, we describe the effects of six fixatives (acidified Lugol's iodine with or without sodium thiosulphate, glutaraldehyde, paraformaldehyde (PFA), formalin and ethanol) on quantitative real-time polymerase chain reaction (qPCR) amplification with Taqman probes. We applied extracted total genomic DNA from four harmful algal species from Danish waters, representing three dinoflagellates (Alexandrium tamarense, Karenia mikimotoi, Karlodinium veneficum and a haptophyte (Prymnesium parvum). The C_q values generated on the qPCR amplification plot were compared to those of an unfixed sample that acted as a control. For all species positive amplifications were achieved from DNA templates from all preserved samples. However, amplification efficiencies between fixatives and species varied. Yet it was found that Lugol's iodine was the most ideal short-term fixative for enumeration of cells by qPCR as well as being the safest to handle. The effect of age on Lugol's iodine fixed samples was also addressed. Samples were fixed and stored at 5 °C in the dark and total genomic DNA extracted after 24 h, 72 h, 1 week, 2 weeks, 1 month and 2 months. Samples remained stable for 1 month for A. tamarense and K. veneficum and 2 months for K. mikimotoi and P. parvum.