Molecular characterization of Cylindrospermopsis raciborskii strains isolated from Portuguese freshwaters

Cylindrospermopsis raciborskii is a toxic bloom forming cyanobacteria that is a common component of the phytoplankton assemblage in temperate freshwaters, as well as in temperate climates. This species is of major concern in public health, due to its known ability to produce toxins, including cylindrospermopsin and paralytic shellfish poisoning toxin (PSP).In this study, M13 PCR fingerprinting, ERIC PCR fingerprinting and amplification of the internal transcribed spacer (ITS) region were used to characterize nine cultured strains of C. raciborskii, sourced from several freshwater lakes and rivers in Portugal, and two other Australian. Strains belonging to other taxa including Microcystis aeruginosa, Aphanizomenon spp., Planktothrix agardhii and Oscillatoria neglecta were also analysed to evaluate the taxonomical potential of the fingerprinting methods.Data obtained from genomic fingerprinting were used to perform hierarchical cluster analysis and demonstrated ability to differentiate strains at intra-specific level. However, the high level of variability prevents their use as an identification tool. ITS amplification displayed intra-specific polymorphism both in number and length of the obtained amplicons, but revealed itself as a good method for strain clustering. The unsuccessful amplification of peptide synthetase (PS) and polyketide synthase (PKS) genes pointed to the inability of Portuguese C. raciborskii strains to produce cylindrospermopsin. HPLC analysis further confirmed this lack of toxicity, since negative results were obtained for cylindrospermopsin and PSP toxins.     

Genetic variability of the invasive cyanobacteria <Emphasis Type="Italic">Cylindrospermopsis raciborskii </Emphasis>from Bir M’cherga reservoir (Tunisia)

Cylindrospermopsis raciborskii is an invasive freshwater cyanobacteria of tropical origin, also found in temperate regions. Due to its known ability to produce potent toxins, such as cylindrospermopsin and the paralytic shellfish poisoning, this species is of major concern from a water quality perspective. This study presents a genetic characterization of four C. raciborskii strains isolated from the Bir M’cherga Tunisian reservoir. The toxicity assessment was investigated via molecular biology tools, which suggested that all the isolated strains were not producing cylindrospermopsin, saxitoxin, or microcystin. This result was further confirmed by HPLC and MALDI-TOF analyses. However, we report for the first time in C. raciborskii the presence of mcyA and mcyE, two segments of the microcystin synthetase mcy cluster. All the strains were identified taxonomically based on the 16S rRNA sequences, and their phylogenetic relationships were assessed using the rpoC1 region. Tunisian strains formed a distinct clade separated from the other African strains.     

Application of real-time PCR in the assessment of the toxic cyanobacterium <Emphasis Type="Italic">Cylindrospermopsis raciborskii</Emphasis> abundance and toxicological potential

Cyanobacteria are prokaryotic photosynthetic microorganisms that pose a serious threat to aquatic environments because they are able to form blooms under eutrophic conditions and produce toxins. Cylindrospermopsis raciborskii is a planktonic heterocystous filamentous cyanobacterium initially assigned to the tropics but currently being found in more temperate regions such as Portugal, the southernmost record for this species in Europe. Cylindrospermopsin originally isolated from C. raciborskii is a cytotoxic alkaloid that affects the liver, kidney, and other organs. It has a great environmental impact associated with cattle mortality and human morbidity. Aiming in monitoring this cyanobacterium and its related toxin, a shallow pond located in the littoral center of Portugal, Vela Lake, used for agriculture and recreational purposes was monitored for a 2-year period. To accomplish this, we used the real-time PCR methodology in field samples to quantify the variation of specific genetic markers with primers previously described characterizing total cyanobacteria (16S rRNA), C. raciborskii (rpoC1), and cylindrospermopsin synthetase gene (pks). The results report the high abundance of both cyanobacteria and C. raciborskii in Vela Lake, with C. raciborskii representing 0.4% to 58% of the total cyanobacteria population. Cylindrospermopsin synthetase gene was detected in one of the samples. We believe that with the approach developed in this study, it will be possible to monitor C. raciborskii population dynamics and seasonal variation, as well as the potential toxin production in other aquatic environments.     

Toxicity phenotype does not correlate with phylogeny of Cylindrospermopsis raciborskii strains

Cylindrospermopsis raciborskii is a species of freshwater, bloom-forming cyanobacterium. C. raciborskii produces toxins, including cylindrospermopsin (hepatotoxin) and saxitoxin (neurotoxin), although non toxin-producing strains are also observed. In spite of differences in toxicity, C. raciborskii strains comprise a monophyletic group, based upon 16S rRNA gene sequence identities (greater than 99%). We performed phylogenetic analyses; 16S rRNA gene and 16S-23S rRNA gene internally transcribed spacer (ITS-1) sequence comparisons, and genomic DNA restriction fragment length polymorphism (RFLP), resolved by pulsed-field gel electrophoresis (PFGE), of strains of C. raciborskii, obtained mainly from the Australian phylogeographic cluster. Our results showed no correlation between toxic phenotype and phylogenetic association in the Australian strains. Analyses of the 16S rRNA gene and the respective ITS-1 sequences (long L, and short S) showed an independent evolution of each ribosomal operon. The genes putatively involved in the cylindrospermopsin biosynthetic pathway were present in one locus and only in the hepatotoxic strains, demonstrating a common genomic organization for these genes and the absence of mutated or inactivated biosynthetic genes in the non toxic strains. In summary, our results support the hypothesis that the genes involved in toxicity may have been transferred as an island by processes of gene lateral transfer, rather than convergent evolution.     

Elevated co2 has Differential Effects on Five Species of Microalgae from a Subtropical Freshwater Lake: Possible Implications for Phytoplankton Species Composition

Rising atmospheric CO₂ concentrations are predicted to have a significant impact on global phytoplankton populations. Of particular interest in freshwater systems are those species that produce toxins or impact water quality, though evidence for how these species, and many others, will respond is limited. This study investigated the effects of elevated CO₂ (1,000 ppm) relative to current atmospheric CO₂ partial pressures (400 ppm), on growth, cell size, carbon acquisition, and photophysiology of five freshwater phytoplankton species including a toxic cyanophyte, Raphidiopsis raciborskii, from Lake Wivenhoe, Australia. Effects of elevated CO₂ on growth rate varied between species; notably growth rate was considerably higher for Staurastrum sp. and significantly lower for Stichococcus sp. with a trend to lower growth rate for R. raciborskii. Surface area to volume ratio was significantly lower with elevated CO₂, for all species except Cyclotella sp. Timing of maximum cell concentrations of those genera studied in monoculture occurred in the lake in order of CO₂ affinity when free CO₂ concentrations dropped below air equilibrium. The results presented here suggest that as atmospheric levels of CO₂ rise, R. raciborskii may become less of a problem to water quality, while some species of chlorophytes may become more dominant. This has implications for stakeholders of many freshwater systems.     

Genetic characterisation of Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria) isolates from Africa and Europe

The invasive cyanobacterium Cylindrospermopsis raciborskii is increasingly spreading in temperate freshwater habitats worldwide and is of major concern due to its ability to produce potent toxins. It is, therefore, important to understand the mechanisms behind the dispersal of this species. Different hypotheses have been proposed to explain the phylogeography and mechanisms underlying the recent expansion of C. raciborskii into temperate latitudes, but there is still no conclusive evidence whether the obvious ecological success of C. raciborskii is due to selection mechanisms, physiological tolerance, climatic change or radiation after the last ice age. In the present study, new isolates of C. raciborskii from Europe and Africa were genetically characterised by sequencing the ITS1, PC-IGS, nifH and rpoC1 genes and compared to corresponding sequences of C. raciborskii available in GenBank in order to test different phylogeographical hypotheses. The strains were also morphologically examined and screened for production of the hepatotoxic cylindrospermopsin (CYN). We clearly demonstrate a variation among the populations of C. raciborskii from different geographical regions. The phylogenetic analyses revealed a clustering of the strains due to geographic origin. The ITS1 and nifH genes separated into American, European and Australian–African groups, whereas the PC-IGS and rpoC1 separated into American and European/Australian/African groups. An analysis of concatenated data supported the division into American, European and African/Australian groups, and even indicated a subdivision into an African and an Australian group. Our findings do not strongly support any of the existing hypotheses on the phylogeography of C. raciborskii, and most likely a combination of these hypotheses is the best approach to understand the evolution and dispersal of this species.     

Sublethal responses in Melanoides tuberculata following exposure to Cylindrospermopsis raciborskii containing cylindrospermopsin

Sublethal effects in the aquatic snail Melanoides tuberculata were examined during exposure to whole cell extracts of Cylindrospermopsis raciborskii and live C. raciborskii cultures, containing varying concentrations of algal cells, cellular debris, and the blue-green algal toxin, cylindrospermopsin (CYN). Exposure to whole cell extracts or live algal cultures did not result in significant changes in adult snail behaviour or relative growth rates. However, clear changes in the number of hatchlings released from parent snails were observed. Exposure to whole cell extracts containing ≥200 μg L⁻¹ extracellular CYN resulted in an increase in the number of hatchlings. In contrast, decreases in hatchling number were recorded from treatments containing ≥200 μg L⁻¹ CYN during exposures to live C. raciborskii cultures, compared with controls. This suggests that CYN may be more toxic to grazing invertebrates if present in the intracellular form. Since CYN is a protein synthesis inhibitor, it is possible that CYN may be especially toxic to rapidly developing tissues such as snail embryos. This may also explain the lack of effects observed in adult snails.     

Genetic and eco-physiological differences of South American Cylindrospermopsis raciborskii isolates support the hypothesis of multiple ecotypes

The global distribution of the toxic cyanobacterium Cylindrospermopsis raciborskii has recently increased, and it has now been identified in tropical, subtropical and temperate freshwater bodies. The mechanisms underlying its success and expansion are still unknown. Several hypotheses have been proposed, including climate change, natural selection and physiological tolerance to different environmental conditions. In this study, we determined the phenotypic and genotypic characteristics of two recently isolated South American strains of C. raciborskii obtained from Uruguay. We analyzed the morphology, growth preferences, tolerance to low temperature (14 °C) and toxin production of the strains and performed phylogenetic analyses based on the ITS and nifH gene sequences. Both isolates showed significantly different morphology and growth behavior under different light intensities and phosphate supply. When genetic differences were assessed by BOX PCR, cluster analyses revealed that they could also be distinguished genotypically and were clearly distinct from C. raciborskii isolated from other continents. Phylogenetic analysis showed that the Uruguayan strains were closely affiliated to other C. raciborskii isolated from the Americas, especially to those from Brazil. Similar to previous studies, we found three solid clusters (Africa-Australia, Europe and America) according to the geographical origin of the isolates. Interestingly, based on nifH sequences, subclusters were identified in American populations indicating an early spread of the species within the continent. We propose that phenotypic and genetic variability of C. raciborskii populations is linked to the existence of different ecotypes whose success is subject to the local environmental conditions.     

Constitutive toxin production under various nitrogen and phosphorus regimes of three ecotypes of Cylindrospermopsis raciborskii ((Wołoszyńska) Seenayya et Subba Raju)

Cylindrospermopsis raciborskii is a global invasive cyanobacterium, with some ecotypes (i.e. strains) producing the toxin cylindrospermopsin, CYN. Multiple ecotypes can co-exist, complicating prediction of toxin concentrations based on cell concentrations. This study examined the growth response and toxin production of three Australian ecotypes of C. raciborskii, two toxic (CS-505, CS-506) and one non-toxic (CS-510), to a range of nitrogen (N) and phosphorus (P) concentrations. CYN cell quota was constant under all N:P ratios and concentration conditions, indicative of a constitutive response, yet the CYN cell quota was 6-fold higher in CS-506 compared to CS-505. The ecotypes differed in response to dissolved N depletion: there was a 4-fold difference in the number of cells heterocyst mL⁻¹ between CS-505 and CS-510, while CS-506 did not produce any heterocysts and was unable to grow in N deplete conditions. Growth rates were lower for all ecotypes as [P] increased, indicative of a species with a strategy of P storage rather than increased growth. Presumably this is an adaptation to low and fluctuating P conditions. However, the negative effect of increasing [P] on growth is surprising. In contrast, increasing [N] resulted in higher growth rates across ecotypes. This study highlights the importance of understanding differences in growth and toxin production between ecotypes in response to environmental conditions in order to more effectively predict blooms and toxin yields.     

High local trait variability in a globally invasive cyanobacterium

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FIRST REPORT OF THE CYANOTOXIN ANATOXIN‐A FROM APHANIZOMENON ISSATSCHENKOI (CYANOBACTERIA)1

The taxonomy and toxicity of a single‐filament isolate from a filamentous cyanobacterial bloom in Lake Hakanoa (New Zealand) were examined by microscopy and liquid chromatography–mass spectrometry. Based on a morphological examination of environmental and cultured material, strain CAWBG02 was identified as Raphidiopsis mediterranea Skuja; however, subsequent phylogenetic analysis of the 16S rRNA gene sequence demonstrated that CAWBG02 was most likely to be a single culture of Aphanizomenon issatschenkoi (Usacev) Proshkina‐Lavrenko. Toxin testing confirmed that the original bloom and A. issatschenkoi isolate produced anatoxin‐a but did not produce homoanatoxin‐a or any cylindrospermopsins, saxitoxins, or microcystins. Despite the absence of cylindrospermopsin production, genes implicated in the biosynthesis of cylindrospermopsin were successfully amplified from A. issatschenkoi strain CAWBG02. To our knowledge, this is the first confirmation of an anatoxin‐a‐producing species in the Southern Hemisphere and the first report of anatoxin‐a production by A. issatschenkoi.     

The rise of potentially toxin producing cyanobacteria in Lake Naivasha,Great African Rift Valley,Kenya

Lake Naivasha, an important inland water ecosystem and a crucial freshwater resource in the Great African Rift Valley, has displayed clear signals of degradation in recent decades. We studied the phytoplankton composition and biomass levels in the period 2001–2013 and noted a progressive increase in the occurrence of potentially toxic cyanobacteria. Analyses for the presence of cyanotoxins such as microcystins (MC), cylindrospermopsin (CYN) and anatoxin-a (ATX-a) were carried out on samples collected in 2008–2013. Among the cyanotoxins tested, low concentrations of MC were detected in the lake. This is the first record of the occurrence of MC in Lake Naivasha. For the first time, molecular phylogenetic investigations of field clones of cyanobacteria from Lake Naivasha were carried out to establish the taxa of the dominant species. Amplification of the aminotrasferase (AMT) domain responsible for cyanotoxin production confirmed the presence of the mcyE gene belonging to the microcystin synthesis gene cluster in field samples containing Microcystis and Planktothrix species. These findings suggest that toxin producing cyanobacteria could become a threat to users of this over-exploited tropical lake in the near future.     

Interspecific allelopathy in cyanobacteria: Cylindrospermopsin and Cylindrospermopsis raciborskii effect on the growth and metabolism of Microcystis aeruginosa

The biological role of cyanobacteria secondary metabolites is relatively unknown although several possible hypotheses have been discussed. In the following study the effect of cylindrospermopsin (CYN) and metabolites of non-CYN producing Cylindrospermopsis raciborskii strain on growth, alkaline phosphatase (ALP) activity and microcystin-LR (MC-LR) production in Microcystis aeruginosa was evaluated. Higher concentrations of CYN (10 and 50 μg L⁻¹) induced toxicity effects demonstrated by significant growth inhibition and M. aeruginosa cell necrosis. Lower concentrations of CYN (1 and 5 μg L⁻¹) slightly decreased growth rates but significantly up-regulated ALP activity. Moreover, under all studied CYN concentrations MC-LR production strongly decreased. Spent C. raciborskii medium mimicked the CYN action by inducing strong inhibition of M. aeruginosa growth and MC-LR production and through up-regulation of ALP activity. On the other hand, spent M. aeruginosa medium did not affect C. raciborskii growth and no alterations in ALP activity were observed. Co-culturing of these two species resulted in an increase of C. raciborskii contribution at the expense of M. aeruginosa. From the results we conclude that CYN can be involved in interspecific competition in cyanobacteria and that non-CYN producing C. raciborskii strains may produce a hitherto unknown bioactive compound(s) which can mimic CYN action.     

Cylindrospermopsin is not degraded by co-occurring natural bacterial communities during a 40-day study

Biodegradation of cylindrospermopsin produced by Aphanizomenon ovalisporum UAM 290 was studied. In the 40-day degradation experiment conducted, bacterial communities from two waterbodies with and without previous exposure to the toxin were used. Further, and in order to study the potential effect of other organic substrates on the degradation of cylindrospermopsin, three different sources of cylindrospermopsin were used: toxic extracts obtained by methanolic extraction and by ultrasonication in water with 5% formic acid and 0.9% NaCl and toxin naturally present in the spent media of an Aphanizomenon ovalisporum culture. Despite active growth of the bacterial population and consumption of DOC in presence of the toxic extracts, no degradation of cylindrospermopsin could be observed during the 40-day period. Considering that cylindrospermopsin is abundant in the extracellular fraction and that photodegradation in the field seems to be limited, a lack of efficient biodegradation as observed in our study could be of greatest importance and further explain the accumulation of this toxin in the dissolved fraction of the waterbodies investigated.     

Domination of invasive Nostocales (Cyanoprokaryota) at 52°N latitude

Cylindrospermopsis raciborskii and Sphaerospermopsis aphanizomenoides are increasingly widespread cyanobacterial species, considered invasive. However, they have never been found to dominate the phytoplankton in the northern part of the invaded area. The aim of our study was to expand the understanding of the invasion process in Nostocales, by answering the question which environmental conditions enabled the first ever noted bloom incident of C. raciborskii and S. aphanizomenoides in the northern part of their current occurrence ranges. We examined the population dynamics of the two invasive cyanobacteria and the abiotic conditions in which their blooms developed in a reservoir at 52°N. We also examined the phytoplankton community diversity and composition before and during the invasive species blooms. As shown by redundancy analysis, the competitive advantage of S. aphanizomenoides depended strongly on high water temperature and high concentration of phosphates, and was positively related to other summer conditions (low water transparency, high ammonium nitrogen concentration). C. raciborskii biomass was not related to temperature and phosphates, but we argue that high water temperature in the first half of July enabled abundant akinete germination, and pulsed phosphorus availability synergized to bring about a later bloom. Both the invasive species co‐occurred with the native cyanobacterium Aphanizomenon gracile, but C. raciborskii bloomed after the native species domination period. The results of the study corroborate the high competitiveness of S. aphanizomenoides in hot, nutrient‐rich conditions, and the adaptation of the invaders to the environment in the invaded regions, inter alia by the overwintering mechanism of numerous akinete differentiation. Our results also contradict the assumption that the phytoplankton diversity index would decrease after colonization of freshwater bodies by invasive cyanoprokaryotes.     

Characterization of the Gene Cluster Responsible for Cylindrospermopsin Biosynthesis

Toxic cyanobacterial blooms cause economic losses and pose significant public health threats on a global scale. Characterization of the gene cluster for the biosynthesis of the cyanobacterial toxin cylindrospermopsin (cyr) in Cylindrospermopsis raciborskii AWT205 is described, and the complete biosynthetic pathway is proposed. The cyr gene cluster spans 43 kb and is comprised of 15 open reading frames containing genes required for the biosynthesis, regulation, and export of the toxin. Biosynthesis is initiated via an amidinotransfer onto glycine followed by five polyketide extensions and subsequent reductions, and rings are formed via Michael additions in a stepwise manner. The uracil ring is formed by a novel pyrimidine biosynthesis mechanism and tailoring reactions, including sulfation and hydroxylation that complete biosynthesis. These findings enable the design of toxic strain-specific probes and allow the future study of the regulation and biological role of cylindrospermopsin.     

FACTORS RELATED TO THE DOMINANCE OF CYLINDROSPERMOPSIS RACIBORSKII (CYANOBACTERIA) IN A SHALLOW POND IN NORTHERN TAIWAN1

Seasonal succession of phytoplankton was investigated in a shallow pond in northern Taiwan from August 2009 to January 2011, with particular reference to the dynamics of Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju. The abundances of the representative species in the pond increased during high‐temperature seasons, whereas only C. raciborskii became dominant in the pond from summer to autumn in both 2009 and 2010. The high shade tolerance of C. raciborskii was likely one of the factors that enabled the cyanobacterium to grow during the summer when the transparency was low. Moreover, the heterocyst production of C. raciborskii was enhanced during summer when the concentration of dissolved inorganic nitrogen was low, implying that nitrogen fixation also played an important role in supporting the growth of C. raciborskii. Autumnal rainfall was a critical factor in the collapse of C. raciborskii blooms. C. raciborskii formed blooms with relatively small trichomes, whereas larger trichomes dominated during winter. The dependence of the trade‐off between growth rate and trichome size on temperature was assumed to be an adaptation strategy of C. raciborskii.     

Effects of thermal acclimation and photoacclimation on lipophilic pigments in an invasive and a native cyanobacterium of temperate regions

The freshwater cyanobacterium Cylindrospermopsis raciborskii spreads from tropical to temperate regions worldwide. This entails acclimation to varied light and temperature conditions. We studied the thermal and light acclimation of the photosynthetic machinery of C. raciborskii by monitoring alteration of the chlorophyll a and carotenoid content in German strains of C. raciborskii, in African and Australian strains of C. raciborskii, and in German strains of Aphanizomenon gracile, a native cyanobacterium belonging to the same order (Nostocales). Our results showed that temperate and tropical C. raciborskii strains did not differ in pigment acclimation to light and temperature. In contrast, the ratio of photoprotective carotenoids (namely the carotenoid glycoside 4-hydroxymyxol glycoside [aphanizophyll]) to chlorophyll a increased significantly more in C. raciborskii in comparison with A. gracile (1) with decreasing temperatures from 20 to 10°C and a moderate light intensity of 80?µmol photons m^?2?s^?1 and (2) with increasing light intensities at a suboptimal temperature of 15°C, compared to 20°C. We conclude that below 20°C photoinhibition is avoided by greater photoprotection in the invasive species C. raciborskii compared to the native species A. gracile.     

Variations in carbon-to-phosphorus ratios of two Australian strains of Cylindrospermopsis raciborskii

The toxic cyanobacterium Cylindrospermopsis raciborskii can form large blooms in freshwater systems, causing water quality problems. The availability of the essential macronutrient phosphorus (P), has a big impact on bloom formation but the variation in physiological response of different strains of C. raciborskii to available P has not previously been examined. This study investigated the carbon:phosphorus (C:P) ratio of two toxic Australian strains of C. raciborskii, AWT205 and NPD, under a range of P concentrations in batch and continuous cultures. P was added as a single dose to batch cultures and in continuous cultures at P concentrations of 0.032, 0.16, 0.64 and 16 μmol P l^?1. Cellular carbon and phosphorus content of both strains increased under P-limited conditions (0 μmol P l^?1 addition) with zero growth. Strain NPD had a lower C:P ratio (34:1) than AWT205 (150:1) indicating higher P storage capacity, and strain NPD survived P-limited conditions for longer. There was no significant difference in exponential growth rates (0.2 d^?1, P ≥ 0.5) under all P concentrations for both strains, with the exception of no P, demonstrating non-P-limited growth even at the lowest concentration (0.032 µmol P l^?1) and no increase in growth rate with additional P. ³³P uptake measurements were used to show that these strains both have very low half saturation constants (K_s = 0.02 μmol P l^?1) compared with other phytoplankton and strains of C. raciborskii. This is indicative of high uptake affinities and suggests that these strains are highly adapted to a low P supply. Overall the results of this study are consistent with the P strategy of storage prioritization over growth rate, and demonstrate differences between the strains in the C:P ratio under P-limitation, indicating variation in P storage.     

PULSES OF PHOSPHATE PROMOTE DOMINANCE OF THE TOXIC CYANOPHYTE CYLINDROSPERMOPSIS RACIBORSKII IN A SUBTROPICAL WATER RESERVOIR1

The role of dissolved inorganic phosphorus (DIP) in promoting dominance of the toxic nitrogen (N)‐fixing cyanobacterium Cylindrospermopsis raciborskii (Wo?osz.) Seenayya et Subba Raju was examined in a subtropical water reservoir, Lake Samsonvale (=North Pine reservoir). A novel in situ bioassay approach, using dialysis tubing rather than bottles or bags, was used to determine the change in C. raciborskii dominance with daily additions of DIP. A statistically significant increase in dominance of C. raciborskii was observed when DIP was added at two concentrations (0.32 μM and 16 μM) in a daily pulse over a 4 d period in three separate experiments in the summer of 2006/2007. There was an increase in both C. raciborskii cell concentrations and biovolume in two DIP treatments, but not in the ammoniacal N + DIP treatment. In addition, overall phytoplankton cell concentrations increased with DIP addition, indicating that Lake Samsonvale was DIP limited at the time of experiments. Given the bioassay response, it is likely that dominance of C. raciborskii could increase in Lake Samsonvale with periodic injections of DIP such as inflow events.