Non-toxic Strains Research Articles

Effects of Cylindrospermopsis raciborskii strains (Woloszynska, 1912) Senayya & Subba Raju on the mobility of Daphnia laevis (Cladocera, Daphniidae).

Cylindrospermopsis raciborskii is a cyanobacterium distributed worldwide that is known to produce cyanotoxins. Some of the Brazilian strains can produce saxitoxins (STXs), which are classified as neurotoxins and can paralyze cladocerans .Daphnia laevis is a cladoceran with a wide distribution in the Americas and has been studied as a possible test-organism in toxicity bioassays. The present work tested the acute effect on D laevis mobility when fed a saxitoxin-producing (STX and neoSTX) C. raciborskii strain, CYRF-01, and compared the results with the effects of a non-toxic strain (NPCS-1). Neonates (6-24 hours after birth) were exposed to concentrations of C. raciborskii varying from 102 to 106 cells·mL-1 of each strain for up to three hours. The cladocerans were then transferred to a medium without toxic filaments for 24 hours. Only the organisms exposed to the STX-producing strain showed signs of the immobilization of swimming movements, confirming the effects of the toxins. There was a linear correlation between the time required to induce stopping the swimming movement, with a shorter time to needed to induce immobilization at a higher the concentration; this correlation was inverse to the time required to recover the swimming movements (longer at higher concentrations, p < 0.1). D. laevis is a tropical and subtropical species with great potential for use in toxicity tests for the detection of STXs, despite being native to and found in a great array of freshwater bodies. This is the first assay testing STX-producing and non-producing C. raciborskii strains on D. laevis, species that are both found in Brazilian ecosystems.

Photosynthetic characteristics of two Cylindrospermopsis raciborskii strains differing in their toxicity.

We studied the growth and photosynthetic characteristics of a toxic (CS506) and a nontoxic strain (CS509) of the bloom-forming cyanobacterium Cylindrospermopsis raciborskii grown under identical experimental conditions. When exposed to light-saturating growth conditions (100 μmol photons · m(-2) ·s(-1) ), values for maximal photosynthetic capacity (Pmax ) and maximum quantum yield (Fv /Fm ) indicated that both strains had an equal ability to process captured photons and deliver them to PSII reaction centers. However, CS506 grew faster than CS509. This was consistent with its higher light requirement for saturation of photosynthesis (Ik ). Greater shade tolerance of CS509 was indicated by its higher ability to harvest light (α), lower photosynthetic light compensation point (Ic ), and higher chlorophyll a to biovolume ratio. Strain-specific differences were found in relation to non-photochemical quenching, effective absorption cross-sectional area of PSIIα-centers (σPSIIα), and the antenna connectivity parameter of PSIIα (Jcon PSIIα). These findings highlighted differences in the transfer of excitation from phycobilisome/PSII to PSI, on the dependence on different pigments for light harvesting and on the functioning of the PSII reaction centers between the two strains. The results of this study showed that both performance and composition of the photosynthetic apparatus are different between these strains, though with only two strains examined we cannot attribute the performance of strain 506 to its ability to produce cylindrospermopsins. The emphasis on a strain-specific light adaptation/acclimation is crucial to our understanding of how different light conditions (both quantity and quality) can trigger the occurrence of different C.raciborskii strains and control their competition and/or dominance in natural ecosystems.

A Single Nucleotide Polymorphism in the Dopamine Receptor D2 Gene May Be Informative for Resistance to Fescue Toxicosis in Angus-Based Cattle

Fescue toxicosis (FT) reduces beef animal growth and fertility. Animals afflicted with FT typically have decreased circulating prolactin concentrations and thicker summer hair coats. Preliminary experiments examined the informativeness of a novel Dopamine Receptor 2 (DRD2) G/A SNP for resistance to FT. Steers grazed tall fescue containing a toxic (E+) or non-toxic (NTE) strain of endophyte. Decreased serum prolactin concentrations were observed in GG steers in May compared to AA steers when grazing E+ pastures (P < 0.02). In a second study, GG steers had decreased prolactin concentrations (P = 0.004) and increased hair coat scores (P = 0.01) relative to AA steers when grazing E+ pastures. Allele and genotypic frequencies were different (P = 0.016 and 0.026, respectively) between spring-calving and fall-calving herds grazing E+ pastures, such that the A allele and the AA genotype were more prevalent in spring-calving herds, suggesting active selection for the A allele. Regardless of calving season, AA heifers tended toward fewer days to first calf (733.6 ± 4.4 d) than did GG heifers (756.6 ± 9.2 days; P = 0.055). These results suggest that the DRD2 SNP may have use in selecting animals resistant to FT.

Development and application of a multiplex qPCR technique to detect multiple microcystin-producing cyanobacterial genera in a Canadian freshwater lake

The emergence and persistence of complex blooms comprising multiple toxigenic cyanobacteria genera pose significant challenges for water quality management worldwide. The co-occurrence of morphologically indistinguishable toxic and non-toxic strains makes monitoring and control of these noxious organisms particularly challenging. Conventional monitoring approaches are not only incapable of discriminating toxic from non-toxic strains but also have proven to be less sensitive and specific. In this study, a multiplex quantitative real-time polymerase chain reaction (qPCR) approach was developed and tested for its sensitivity and specificity at detecting, differentiating and estimating potentially toxic Anabaena, Microcystis and Planktothrix genotype compositions in environmental samples. The oligonucleotide primers and probes utilized were designed to target portions of the microcystin synthetase (mcy) E gene that encode synthesis of the unique 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (ADDA) moiety of microcystins in the three target genera. Laboratory evaluation showed the developed assay to be highly sensitive and specific at detecting and quantifying targeted genera. Indeed, the assay standards for the Anabaena, Microcystis and Planktothrix reactions attained efficiencies above 90 %, with coefficients of determination consistently above 0.99. Analysis of water samples from Missisquoi Bay, Quebec, Canada, resulted in successful detection and quantification of target toxigenic cyanobacteria even when cell numbers were below the detection limit for the conventional microscopy methods. Furthermore, toxigenic Microcystis spp. were found to be the main putative microcystin-producing cyanobacteria in the study lake. The qPCR technique developed in this study therefore offers simultaneous detection, differentiation and quantification of multiple toxigenic cyanobacteria that otherwise cannot be accomplished by current monitoring approaches.

Identification and quantification of toxic and nontoxic strains of the harmful dinoflagellate Alexandrium tamarense using fluorescence in situ hybridization and flow cytometry

The co‐occurrence of morphologically identical toxic and nontoxic ribotypes of the biotoxin producing marine dinoflagellate Alexandrium tamarense presents a significant problem for its identification and enumeration, particularly in a regulatory monitoring context. To address this, we have developed a fluorescence in situ hybridization‐flow cytometry (FISH‐FC)—based method of cell identification and enumeration. This employed the taxa specific oligonucleotide probes TamToxC and TamA to fluorescently label (with the fluorochromes CY.3 and FITC) Group I (toxic) and Group III (nontoxic) A. tamarense ribotypes, respectively. Detection was by fluorescence activated flow cytometric analysis. The FISH‐FC method allowed effective discrimination between laboratory cultures of Group I and Group III ribotypes, with toxic and nontoxic cells creating distinct, easily identifiable, clusters in a flow cytometer bi‐plot of side scatter (SSC) versus the green (FL1) fluorescence detection channel. Comparison of estimates of cell abundance obtained by the FISH‐FC technique with those obtained by microscopy (Sedgwick Rafter technique) showed no statistically significant difference across a range of concentrations. Subsequently, the methodology was successfully applied on natural seawater samples, spiked with known concentrations of toxic and nontoxic A. tamarense cells at environmentally relevant concentrations.

Construction of Genome Library for the Toxic Gene of &lt;i&gt;Alexandium minutum&lt;/i&gt;

Genome library of toxic Alexandium minutum were constructed. A 1014bp DNA reconstruction fragment was obtained and PCR method testified that this fragment had strong amplification signal in toxic strains while no signal in non-toxic strains. The external sequence of the DNA fragment was analyzed by inverse PCR method, and a 240bp nucleotide sequence which translated into proteins aminophenol was Methionine Aminopeptidase (MAP). The sequence of aminophenol was BLAST in NCBI, and found that it had 97% similarity with Alexandium fundyense. This sequence was exact uniform with the reported sequence in the coding region, but there were three bases mutated in non-coding region behind stop codon. Probably, in the course of growth metabolism of Alexandium minutum, MAP executed different physiological functions and controlled the production of the toxin. In the meantime, map gene induced encoding products of the related poisonous gene, and enabled it has active expressions to starting toxin synthesis.

The cyanobacterium Cylindrospermopsis raciborskii is facilitated by copepod selective grazing

Blooms of the toxin-producing cyanobacterium Cylindrospermopsis raciborskii occur in tropical and subtropical lakes during spring-summer but the mechanisms behind bloom formation are unclear. This study tests the hypothesis that C. raciborskii accumulations in freshwater systems are facilitated by selective copepod grazing. Prey selection was examined in a series of experiments with C. raciborskii and the green alga, Chlamydomonas reinhardtii, as well as within natural phytoplankton assemblages. Clearance rates of the copepod Boeckella sp. on a C. raciborskii diet were 2–4 times lower than that of a common cladoceran Ceriodaphnia sp. when both grazers had prey choice. More C. raciborskii was cleared by Boeckella sp. when in mixed natural phytoplankton assemblages, but the clearance rate declined when nutrient replete C. reinhardtii was added, demonstrating that when alternate “high quality” algae were present, so did C. raciborskii consumption. The clearance rates of Boeckella sp. on two toxic C. raciborskii strains were significantly lower than on a non-toxic strain, and on C. raciborskii with low cellular P content. When we tested the grazing preference of a copepod dominated mixed zooplankton community on C. raciborskii during the early bloom period, clearance rates were relatively low (0.05–0.20mlindividual−1h−1), and decreased significantly as the proportion of C. raciborskii increased above 5%. These results suggest that C. raciborskii persistence could be promoted by copepods preferentially grazing on other algae, with significant loss of top-down control as C. raciborskii abundance increases.

Whole-Genome Sequence of Microcystis aeruginosa TAIHU98, a Nontoxic Bloom-Forming Strain Isolated from Taihu Lake, China

Microcystis aeruginosa is a dominant bloom-forming cyanobacterium in many freshwater lakes. This report describes the first whole-genome sequence of the nontoxic strain of M. aeruginosa TAIHU98, which was isolated from Taihu Lake in eastern China.

Susceptibility of hamsters to Clostridium difficile isolates of differing toxinotype.

Clostridium difficile is the most commonly associated cause of antibiotic associated disease (AAD), which caused ∼21,000 cases of AAD in 2011 in the U.K. alone. The golden Syrian hamster model of CDI is an acute model displaying many of the clinical features of C. difficile disease. Using this model we characterised three clinical strains of C. difficile, all differing in toxinotype; CD1342 (PaLoc negative), M68 (toxinotype VIII) & BI-7 (toxinotype III). The naturally occurring non-toxic strain colonised all hamsters within 1-day post challenge (d.p.c.) with high-levels of spores being shed in the faeces of animals that appeared well throughout the entire experiment. However, some changes including increased neutrophil influx and unclotted red blood cells were observed at early time points despite the fact that the known C. difficile toxins (TcdA, TcdB and CDT) are absent from the genome. In contrast, hamsters challenged with strain M68 resulted in a 45% mortality rate, with those that survived challenge remaining highly colonised. It is currently unclear why some hamsters survive infection, as bacterial & toxin levels and histology scores were similar to those culled at a similar time-point. Hamsters challenged with strain BI-7 resulted in a rapid fatal infection in 100% of the hamsters approximately 26 hr post challenge. Severe caecal pathology, including transmural neutrophil infiltrates and extensive submucosal damage correlated with high levels of toxin measured in gut filtrates ex vivo. These data describes the infection kinetics and disease outcomes of 3 clinical C. difficile isolates differing in toxin carriage and provides additional insights to the role of each toxin in disease progression.

PCR primers for selective detection of intra-species variations in the bloom-forming cyanobacterium, Microcystis

Members of the cyanobacterial genus Microcystis commonly form blooms in eutrophic freshwater systems, and some produce cyclic heptapeptide hepatotoxins called microcystins, thereby often causing serious water management problems. Microcystis species were unified into the single Microcystis aeruginosa classification based on 16S rRNA gene sequences and DNA–DNA re-association experiments; however, the morphological features of the organisms differ in different culturing conditions. Here, we describe a new real-time quantitative PCR (qPCR) method of determining Microcystis intradiversity using the SYBR Green I assay. We analyzed 71 Microcystis 16S-23S rDNA internal transcribed spacer region (16S-23S ITS) sequences, designed three group-specific PCR primers that successfully selected a morphologically M. wesenbergii-like non-toxic group (Group-3), and differentiated between M. viridis-like toxic group (Group-4) and M. aeruginosa-like Group-1 organisms including toxic and non-toxic Microcystis strains. The primers covered 76% of the Microcystis 16S-23S ITS regions from all over the world (six continents) included in GenBank. We constructed a mixed culture with representative Microcystis strains from each group, and estimated their cell densities by qPCR over 7 weeks. Group-1 and Group-3 grew exponentially for 4 weeks; however, the growth of Group-4 declined after 2 weeks, revealing different growth properties for the Microcystis groups in the mixed culture. Finally, we applied this method to natural Microcystis blooms at four freshwater sites, and found the dominance of Group-1 in three blooms and of Group-3 in one bloom, thereby showing the geographically uneven distribution of Microcystis genotypes. The developed qPCR technique targeting the 16S-23S ITS region is both rapid and simple and is useful for selective quantification of group variations among sympatric Microcystis genotypes, such as in mixed cultures and the natural environment.

Evaluation of the MIDTAL microarray chip for monitoring toxic microalgae in the Orkney Islands, U.K.

Harmful or nuisance algal blooms can cause economic damage to fisheries and tourism. Additionally, toxins produced by harmful algae and ingested via contaminated shellfish can be potentially fatal to humans. The seas around the Orkney Islands, UK currently hold a number of toxic algal species which cause shellfishery closures in most years. Extensive and costly monitoring programs are carried out to detect harmful microalgae before they reach action levels. However, the ability to distinguish between toxic and non-toxic strains of some algae is not possible using these methods. The microarrays for the detection of toxic algae (MIDTAL) microarray contains rRNA probes for toxic algal species/strains which have been adapted and optimized for microarray use. In order to investigate the use of the chip for monitoring in the Orkney Islands, samples were collected between 2009 and 2011 from Brings Deep, Scapa Flow, Orkney Islands, UK; RNA was extracted and hybridized with generation 2 and 3.1 of the chip. The data were then compared to cell counts performed under light microscopy and in the case of Alexandrium tamarense to qPCR data targeting the saxitoxin gene and the LSU-rRNA gene. A good agreement between cell numbers and microarray signal was found for A. tamarense, Pseudo-nitzschia sp., Dinophysis sp. (r<0.5, for all) in addition to this there the chip successfully detected a large bloom of Karenia mikimotoi (r<0.70) in August and September 2011. Overall, there was good improvement in probe signal between generation 2 and generation 3.1 of the chip with much less variability and more consistent results and better correlation between the probes. The chip performed well for A. tamarense group I signal to cell numbers in calibrations (r>0.9). However, in field samples, this correlation was slightly lower suggesting interactions between all species in the sample may affect signal. Overall, the chip showed it could identify the presence of target species in field samples although some work is needed to improve the quantitative nature of the chip before it would be suitable for monitoring in the Orkney Islands.

Bacillus “next generation” diagnostics: moving from detection toward subtyping and risk-related strain profiling

The highly heterogeneous genus Bacillus comprises the largest species group of endospore forming bacteria. Because of their ubiquitous nature, Bacillus spores can enter food production at several stages resulting in significant economic losses and posing a potential risk to consumers due the capacity of certain Bacillus strains for toxin production. In the past, food microbiological diagnostics was focused on the determination of species using conventional culture-based methods, which are still widely used. However, due to the extreme intra-species diversity found in the genus Bacillus, DNA-based identification and typing methods are gaining increasing importance in routine diagnostics. Several studies showed that certain characteristics are rather strain-dependent than species-specific. Therefore, the challenge for current and future Bacillus diagnostics is not only the efficient and accurate identification on species level but also the development of rapid methods to identify strains with specific characteristics (such as stress resistance or spoilage potential), trace contamination sources, and last but not least discriminate potential hazardous strains from non-toxic strains.

Effect of non-toxic, filamentous cyanobacteria on egg abortion in Daphnia under various thermal conditions

The aim of this study was to examine whether the presence of non-toxic filamentous cyanobacteria can cause egg abortion in Daphnia. To test this hypothesis, we fed females of Daphnia pulicaria with the green alga Scenedesmus obliquus from birth until eggs were laid in the brood chamber. Gravid Daphnia females were then exposed to media containing either S. obliquus or filaments of a non-toxic strain of the cyanobacterium Cylindrospermopsis raciborskii. The females were then observed throughout the whole period of egg development and all events of egg abortion were recorded. Experiments were conducted at three temperatures: 16, 20, and 24°C. In the presence of cyanobacteria, the proportion of aborted eggs ranged from 2.6 to 12.9%, compared with 0–1.8% in the green algae treatment. The number of aborted eggs, but not the number of egg-aborting females, tended to increase at higher temperature. Our data indicate that interference by non-toxic cyanobacterial filaments may affect the reproductive success of Daphnia, not only through suppression of their fecundity, but also via an increase in the number of aborted eggs. Therefore, the reproductive rate of Daphnia based on the fecundity of females may be overestimated in situations where filamentous cyanobacteria are present.

Anatoxin-a-producing Raphidiopsis mediterranea Skuja var. grandis Hill is one ecotype of non-heterocytous Cuspidothrix issatschenkoi (Usačev) Rajaniemi et al. in Japanese lakes

In this study, we sought to clarify the phylogenetic and ecological relationships of anatoxin-a-producing strains of Cuspidothrix issatschenkoi (Usačev) Rajaniemi et al. and Raphidiopsis mediterranea Skuja var. grandis Hill in Japanese lakes. We isolated 60 strains from six water bodies and evaluated their ability to produce anatoxin-a and nitrogenase, and determined the sequences of the 16S–23S rDNA internal transcribed spacer region. The results consistently indicated that all R. mediterranea ver. grandis-like strains were non-heterocytous, anatoxin-a-producing C. issatschenkoi. We also evaluated the distribution of anatoxin-a-producing strains of C. issatschenkoi in 88 water bodies using primers and a probe specifically developed in this study for quantitative PCR. By this method, we detected anatoxin-a-producing strains in 16 water bodies, while by automated rRNA intergenic spacer analysis, C. issatschenkoi was detected in 24 water bodies, indicating that the toxic and non-toxic strains occur in different environments. Although C. issatschenkoi has been classified as the functional group H1, which is tolerant to low nitrogen concentrations, the anatoxin-a-producing strains were detected from water bodies with low TOC/TON because of the lack of nitrogen-fixing ability. These results suggest that different genotypes of C. issatschenkoi have different ecotypes and prefer different environments.

The Newly Described Heterotrophic Dinoflagellate Gyrodinium moestrupii, an Effective Protistan Grazer of Toxic Dinoflagellates

Few protistan grazers feed on toxic dinoflagellates, and low grazing pressure on toxic dinoflagellates allows these dinoflagellates to form red-tide patches. We explored the feeding ecology of the newly described heterotrophic dinoflagellate Gyrodinium moestrupii when it fed on toxic strains of Alexandrium minutum, Alexandrium tamarense, and Karenia brevis and on nontoxic strains of A. tamarense, Prorocentrum minimum, and Scrippsiella trochoidea. Specific growth rates of G. moestrupii feeding on each of these dinoflagellates either increased continuously or became saturated with increasing mean prey concentration. The maximum specific growth rate of G. moestrupii feeding on toxic A. minutum (1.60/d) was higher than that when feeding on nontoxic S. trochoidea (1.50/d) or P. minimum (1.07/d). In addition, the maximum growth rate of G. moestrupii feeding on the toxic strain of A. tamarense (0.68/d) was similar to that when feeding on the nontoxic strain of A. tamarense (0.71/d). Furthermore, the maximum ingestion rate of G. moestrupii on A. minutum (2.6 ng C/grazer/d) was comparable to that of S. trochoidea (3.0 ng C/grazer/d). Additionally, the maximum ingestion rate of G. moestrupii on the toxic strain of A. tamarense (2.1 ng C/grazer/d) was higher than that when feeding on the nontoxic strain of A. tamarense (1.3 ng C/grazer/d). Thus, feeding by G. moestrupii is not suppressed by toxic dinoflagellate prey, suggesting that it is an effective protistan grazer of toxic dinoflagellates.

Evolution of Saxitoxin Synthesis in Cyanobacteria and Dinoflagellates

Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. Saxitoxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX synthesis genes, including all of the genes directly involved in toxin synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxitoxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some evolutionarily related proteins. The biological role of STX is not well understood in either cyanobacteria or dinoflagellates. However, STX production in these two ecologically distinct groups of organisms suggests that this toxin confers a benefit to producers that we do not yet fully understand.

COMPARISON OF RESISTANCE TO LIGHT STRESS IN TOXIC AND NON-TOXIC STRAINS OF MICROCYSTIS AERUGINOSA (CYANOPHYTA)(1).

Blooms of Microcystis aeruginosa (Kützing) Kützing occur frequently in many freshwater ecosystems around the world, but the role of environmental factors in promoting the growth and determining the proportion of toxic and non-toxic strains still requires more investigation. In this study, four strains (toxic CPCC299 & FACHB905 and non-toxic CPCC632 & FACHB315) were exposed to high light (HL) condition, similar to light intensity found at the surface of a bloom, to evaluate their sensitivity to photoinhibition. We also estimated their capacity to recover from this HL stress. For all strains, our results showed an increased inhibition of the photosynthetic activity with HL treatment time. When comparing the extent of photoinhibition between strains, both toxic strains were more resistant to the treatment and recovered completely their photosynthetic capacity after 3 h, while non-toxic strains needed more time to recover. For toxic strains, the rETR under HL was higher compared to the rETR under low light (LL) control condition despite 50% photoinhibition. This suggests that the detrimental effect of high light (HL; up to 2 h) is outweighed by their higher photosynthetic potential. This conclusion did not stand for non-toxic strains, and indicates their preference for LL environment. We also demonstrated that a LL/HL cycle induced a 259% increase in cell yield for a toxic strain and a decrease by 22% for a non-toxic strain. This also indicates that toxic strains have higher tolerance to HL in a fluctuating light environment. Our data demonstrated that difference of sensitivity to HL between strains can modify the competitive outcome between toxic and non-toxic strains and may affect bloom toxicity.

Algal Toxins Alter Copepod Feeding Behavior

Using digital holographic cinematography, we quantify and compare the feeding behavior of free-swimming copepods, Acartia tonsa, on nutritional prey (Storeatula major) to that occurring during exposure to toxic and non-toxic strains of Karenia brevis and Karlodinium veneficum. These two harmful algal species produce polyketide toxins with different modes of action and potency. We distinguish between two different beating modes of the copepod’s feeding appendages–a “sampling beating” that has short durations (<100 ms) and involves little fluid entrainment and a longer duration “grazing beating” that persists up to 1200 ms and generates feeding currents. The durations of both beating modes have log-normal distributions. Without prey, A. tonsa only samples the environment at low frequency. Upon introduction of non-toxic food, it increases its sampling time moderately and the grazing period substantially. On mono algal diets for either of the toxic dinoflagellates, sampling time fraction is high but the grazing is very limited. A. tonsa demonstrates aversion to both toxic algal species. In mixtures of S. major and the neurotoxin producing K. brevis, sampling and grazing diminish rapidly, presumably due to neurological effects of consuming brevetoxins while trying to feed on S. major. In contrast, on mixtures of cytotoxin producing K. veneficum, both behavioral modes persist, indicating that intake of karlotoxins does not immediately inhibit the copepod’s grazing behavior. These findings add critical insight into how these algal toxins may influence the copepod’s feeding behavior, and suggest how some harmful algal species may alter top-down control exerted by grazers like copepods.

Impact of copper exposure on Pseudo-nitzschia spp. physiology and domoic acid production

Microalgae have differing sensitivities to copper toxicity. Some species within the genus Pseudo-nitzschia produce domoic acid (DA), a phycotoxin that has been hypothesised to chelate Cu and ameliorate Cu toxicity to the cells. To better characterise the effect of Cu on Pseudo-nitzschia, a toxic strain of P. multiseries and a non-toxic strain of P. delicatissima were exposed to Cu(II) for 96h (50μgl−1 for P. delicatissima and 50, 100 and 150μgl−1 for P. multiseries). Physiological measurements were performed daily on Pseudo-nitzschia cells using fluorescent probes and flow cytometry to determine the cell density, lipid concentration, chlorophyll autofluorescence, esterase activity, percentage of dead algal cells, and number of living and dead bacteria. Photosynthetic efficiency and O2 consumption and production of cells were also measured using pulse amplitude modulated fluorometry and SDR Oxygen Sensor dish. The DA content was measured using ELISA kits. After 48h of Cu exposure, P. delicatissima mortality increased dramatically whereas P. multiseries survival was unchanged (in comparison to control cells). Cellular esterase activity, chlorophyll autofluorescence, and lipid content significantly increased upon Cu exposure in comparison to control cells (24h for P. delicatissima, up to 96h for P. multiseries). Bacterial concentrations in P. multiseries decreased significantly when exposed to Cu, whereas bacterial concentrations were similar between control and exposed populations of P. delicatissima. DA concentrations in P. multiseries were not modified by Cu exposure. Addition of DA to non-toxic P. delicatissima did not enhance cells survival; hence, extracellular DA does not protect Pseudo-nitzschia spp. against copper toxicity. Results suggested that cells of P. delicatissima are much more sensitive to Cu than P. multiseries. This difference is probably not related to the ability of P. multiseries to produce DA but could be explained by species differences in copper sensitivity, or a difference of bacterial community between the algal species.

The dynamics of toxic Microcystis strains and microcystin production in two hypertrofic South African reservoirs

The South African impoundments of Hartbeespoort and Roodeplaat experience excessive blooms of Microcystis species each year. Microcystins, produced primarily by strains of cyanobacteria belonging to the genera Microcystis, Anabaena and Planktothrix, are harmful cyanobacterial hepatotoxins. These bloom-forming cyanobacteria form toxic and non-toxic strains that co-occur and are visually indistinguishable, but can be identified and quantified molecularly. We described the relationships between microcystin production and the genotypic composition of the Microcystis community involved together with environmental conditions in both the Roodeplaat and Hartbeespoort reservoirs using quantitative real time PCR. DNA copy number of the Microcystis-specific 16S rRNA and toxin biosynthesis genes, mcyE and mcyB, were measured. Planktothrix spp. occurred in both reservoirs during autumn, but no toxin-producing species was present as measured with mcyE specific primers, whereas both toxic and non-toxic strains of Microcystis were recorded in both reservoirs, with Microcystis spp. dominating in the summer months. Water-surface temperature correlated strongly with microcystin concentration, mcyE and mcyB copy number. Microcystin production was associated by temperatures higher than 23°C. This suggests that should current environmental trends persist with surface water temperatures continuing to rise and more and more nutrients continued to be loaded into fresh water systems toxic Microcystis may outgrow non-toxic Microcystis and synthesise even more microcystins.