Dietary exposure to harmful algal bloom (HAB) toxins in the endangered manatee (Trichechus manatus latirostris) and green sea turtle (Chelonia mydas) in Florida,USA

Florida is a hotspot for cyano- and microalgal harmful algal blooms (HABs) with annual red-tide events off-shore and blooms of Lyngbya spp. commonly observed in both marine and freshwater environments. This region also provides extensive foraging habitat for large populations of herbivorous green turtles (Chelonia mydas) and manatees (Trichechus manatus latirostris). The exposure of aquatic organisms to HAB toxins is not well known and whilst acute exposures are better understood, the vulnerability of aquatic animals to chronic exposure from multiple HAB toxins over prolonged periods has rarely been addressed. This study aimed to identify the presence of toxic compounds produced by HAB species commonly found in Florida (brevetoxins, okadaic acid, saxitoxins and Lyngbya toxins) in tissues and gut samples from manatee and green sea turtles that stranded in Florida, USA. Muscle, liver and alimentary tract samples were opportunistically collected from 14 manatees and 13 green turtles that stranded on the Florida shoreline between December 2003 and February 2006. Samples from each animal were assessed for the presence of brevetoxin, okadaic acid, lyngbyatoxin-A and saxitoxin. Nine (64%) manatees and 11 (85%) turtles were found to have been exposed to one or more of the HAB toxins. Okadaic acid and saxitoxin were only found in alimentary tract samples, whereas brevetoxin was more widely distributed. No lyngbyatoxin-A was observed in any tissue samples. The majority of turtles (13) stranded on the Atlantic coast between St. Johns and Monroe counties, with one on the Gulf coast at Bay County, whereas nine manatees were stranded on the Gulf coast between Levy and Lee counties, with the remaining five between Volusia and Brevard counties on the Atlantic coast. This HAB toxin screen has identified that a large proportion of a random sample of turtles and manatees that stranded in Florida in 2003–2006 were exposed to HAB toxins. Most of the concentrations measured were low, and the toxins were directly linked to the death of only three of these animals. However, the presence of these compounds, and in some cases the presence of multiple HAB toxins in individual animals, indicates that turtles and manatees in Florida are exposed to deleterious compounds at sub-lethal levels in their environment, which could ultimately compromise their health.     

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.     

The zebrafish (Danio rerio) embryo as a model system for identification and characterization of developmental toxins from marine and freshwater microalgae

The zebrafish (Danio rerio) embryo has emerged as an important model of vertebrate development. As such, this model system is finding utility in the investigation of toxic agents that inhibit, or otherwise interfere with, developmental processes (i.e. developmental toxins), including compounds that have potential relevance to both human and environmental health, as well as biomedicine. Recently, this system has been applied increasingly to the study of microbial toxins, and more specifically, as an aquatic animal model, has been employed to investigate toxins from marine and freshwater microalgae, including those classified among the so-called "harmful algal blooms" (HABs). We have developed this system for identification and characterization of toxins from cyanobacteria (i.e. "blue-green algae") isolated from the Florida Everglades and other freshwater sources in South and Central Florida. Here we review the use of this system as it has been applied generally to the investigation of toxins from marine and freshwater microalgae, and illustrate this utility as we have applied it to the detection, bioassay-guided fractionation and subsequent characterization of developmental toxins from freshwater cyanobacteria.     

Molecular Characterization of the Toxic Cyanobacterium Cylindrospermopsis raciborskii and Design of a Species-Specific PCR

Cylindrospermopsis raciborskii is a toxic-bloom-forming cyanobacterium that is commonly found in tropical to subtropical climatic regions worldwide, but it is also recognized as a common component of cyanobacterial communities in temperate climates. Genetic profiles of C. raciborskii were examined in 19 cultured isolates originating from geographically diverse regions of Australia and represented by two distinct morphotypes. A 609-bp region of rpoC1, a DNA-dependent RNA polymerase gene, was amplified by PCR from these isolates with cyanobacterium-specific primers. Sequence analysis revealed that all isolates belonged to the same species, including morphotypes with straight or coiled trichomes. Additional rpoC1 gene sequences obtained for a range of cyanobacteria highlighted clustering of C. raciborskii with other heterocyst-producing cyanobacteria (orders Nostocales and Stigonematales). In contrast, randomly amplified polymorphic DNA and short tandemly repeated repetitive sequence profiles revealed a greater level of genetic heterogeneity among C. raciborskii isolates than did rpoC1 gene analysis, and unique band profiles were also found among each of the cyanobacterial genera examined. A PCR test targeting a region of the rpoC1 gene unique to C. raciborskii was developed for the specific identification of C. raciborskii from both purified genomic DNA and environmental samples. The PCR was evaluated with a number of cyanobacterial isolates, but a PCR-positive result was only achieved with C. raciborskii. This method provides an accurate alternative to traditional morphological identification of C. raciborskii.     

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.     

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.     

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.     

Early stages of biofilm succession in a lentic freshwater environment

Initial events of biofilms development and succession were studied in a freshwater environment at Kalpakkam, East Coast of India. Biofilms were developed by suspending Perspex (Plexiglass) panels for 15 days at bimonthly intervals from January 1996 to January 1997. Changes in biofilm thickness, biomass, algal density, chlorophyll a concentration and species composition were monitored. The biofilm thickness, biomass, algal density and chlorophyll a concentration increased with biofilms age and colonization was greater during summer (March, May and July) than other months. The initial colonization was mainly composed of Chlorella vulgaris, Chlorococcum humicolo (green algae), Achnanthes minutissima, Cocconeis scutellum, C. placentula (diatoms) and Chroococcus minutus (cyanobacteria) followed by colonial green algae such as Pediastrum tetras, P. boryanumand Coleochaete scutata, cyanobacteria (Gloeocapsa nigrescens), low profile diatoms (Amphora coffeaeformis, Nitzschia amphibia, and Gomphonema parvulum) and long stalked diatoms (Gomphoneis olivaceumand Gomphonema lanceolatum). After the 10th day, the community consisted of filamentous green algae (Klebshormidium subtile, Oedogonium sp., Stigeoclonium tenue and Ulothrix zonata) and cyanobacteria (Calothrix elenkinii, Oscillatoria tenuis and Phormidium tenue). Based on the percentage composition of different groups in the biofilm, three phases of succession could be identified: the first phase was dominated by green algae, the second by diatoms and the third phase by cyanobacteria. Seasonal variation in species composition was observed but the sequence of colonization was similar throughout the study period.     

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.     

The effects of phosphorus and temperature on the competitive success of an invasive cyanobacterium

Rising lake temperatures and changing nutrient inputs are believed to favour the spread of a toxic invasive cyanobacterium, Cylindrospermopsis raciborskii (Woloszynska) Seenayya and Subba Raju, in temperate lakes. However, most evidence for these hypotheses is observational or based on physiological measurements in monocultures. We lack clear experimental evidence relating temperature and nutrients to the competitive success of C. raciborskii. To address this, we performed a 2 × 2 factorial laboratory experiment to study the dynamics of mixed phytoplankton communities subjected to different levels of temperature and phosphorus over 51 days. We allowed C. raciborskii to compete with ten different species from major taxonomic groups (diatoms, green algae, cryptophytes, and cyanobacteria) typical of temperate lakes, under low and high summer temperatures (25 and 30 °C) at two levels of phosphorus supply (1 and 25 µmol L^?1). Cylindrospermopsis raciborskii dominated the communities and strongly decreased diversity under low-phosphorus conditions, consistent with the hypothesis that it is a good phosphorus competitor. In contrast, it remained extremely rare in high-phosphorus conditions, where fast-growing green algae dominated. Surprisingly, temperature played a negligible role in influencing community composition, suggesting that changes in summer temperature may not be important in determining C. raciborskii’s spread.     

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 (0.05–0.20 ml individual⁻¹ h⁻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.     

Remote,subsurface detection of the algal toxin domoic acid onboard the Environmental Sample Processor: Assay development and field trials

The ability to detect harmful algal bloom (HAB) species and their toxins in real- or near real-time is a critical need for researchers studying HAB/toxin dynamics, as well as for coastal resource managers charged with monitoring bloom populations in order to mitigate their wide ranging impacts. The Environmental Sample Processor (ESP), a robotic electromechanical/fluidic system, was developed for the autonomous, subsurface application of molecular diagnostic tests and has successfully detected several HAB species using DNA probe arrays during field deployments. Since toxin production and thus the potential for public health and ecosystem effects varies considerably in natural phytoplankton populations, the concurrent detection of HAB species and their toxins onboard the ESP is essential. We describe herein the development of methods for extracting the algal toxin domoic acid (DA) from Pseudo-nitzschia cells (extraction efficiency >90%) and testing of samples using a competitive ELISA onboard the ESP. The assay detection limit is in the low ng/mL range (in extract), which corresponds to low ng/L levels of DA in seawater for a 0.5 L sample volume acquired by the ESP. We also report the first in situ detection of both a HAB organism (i.e., Pseudo-nitzschia) and its toxin, domoic acid, via the sequential (within 2–3 h) conduct of species- and toxin-specific assays during ESP deployments in Monterey Bay, CA, USA. Efforts are now underway to further refine the assay and conduct additional calibration exercises with the aim of obtaining more reliable, accurate estimates of bloom toxicity and thus their potential impacts.     

On the control of HAB species using low biosurfactant concentrations

Biosurfactants have been suggested as a method to control harmful algal blooms (HABs), but warrant further and more in-depth investigation. Here we have investigated the algicidal effect of a biosurfactant produced by the bacterium Pseudomonas aeruginosa on five diverse marine and freshwater HAB species that have not been tested previously. These include Alexandrium minutum (Dinophycaee), Karenia brevis (Dinophyceae), Pseudonitzschia sp. (Bacillariophyceae), in marine ecosystems, and Gonyostomum semen (Raphidophyceae) and Microcystis aeruginosa (Cyanophyecae) in freshwater. We examined not only lethal but also sub-lethal effects of the biosurfactant. In addition, the effect of the biosurfactant on Daphnia was tested. Our conclusions were that very low biosurfactant concentrations (5 μg mL⁻¹) decreased both the photosynthesis efficiency and the cell viability and that higher concentrations (50 μg mL⁻¹) had lethal effects in four of the five HAB species tested. The low concentrations employed in this study and the diversity of HAB genera tested suggest that biosurfactants may be used to either control initial algal blooms without causing negative side effect to the ecosystem, or to provoke lethal effects when necessary.     

Cylindrospermopsin and Saxitoxin Synthetase Genes in Cylindrospermopsis raciborskii Strains from Brazilian Freshwater

The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX), while cylindrospermopsin (CYN), which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene sequences were obtained for all strains, while cyrB and cyrC gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify cyrJ gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of C. raciborskii strains with high bootstrap support. In addition, fragments of sxtA, sxtB and sxtI genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of cyr genes in South American strains of C. raciborskii and the presence of sxt and cyr genes in a single C. raciborskii strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of C. raciborskii and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins.     

Harmful phytoplankton diversity and dynamics in an upwelling region (Sagres,SW Portugal) revealed by ribosomal RNA microarray combined with microscopy

The study region in Sagres, SW Portugal, is subject to natural eutrophication of coastal waters by wind-driven upwelling, which stimulates high primary productivity facilitating the recent economic expansion of bivalve aquaculture in the region. However, this economic activity is threatened by harmful algal blooms (HAB) caused by the diatoms Pseudo-nitzschia spp., Dinophysis spp. and other HAB dinoflagellates, all of which can produce toxins, that can induce Amnesic Shellfish Poisoning (ASP), Diarrhetic Shellfish Poisoning (DSP) and Paralytic Shellfish Poisoning (PSP). This study couples traditional microscopy with 18S/28S rRNA microarray to improve the detection of HAB species and investigates the relation between HAB and the specific oceanographic conditions in the region. Good agreement was obtained between microscopy and microarray data for diatoms of genus Pseudo-nitzschia and dinoflagellates Dinophysis spp., Gymnodinium catenatum and raphidophyte Heterosigma akashiwo, with less effective results for Prorocentrum. Microarray provided detection of flagellates Prymnesium spp., Pseudochattonella spp., Chloromorum toxicum and the important HAB dinoflagellates of the genera Alexandrium and Azadinium, with the latter being one of the first records from the study region. Seasonality and upwelling induced by northerly winds were found to be the driving forces of HAB development, with Pseudo-nitzschia spp. causing the risk of ASP during spring and summer upwelling season, and dinoflagellates causing the risk of DSP and PSP during upwelling relaxation, mainly in summer and autumn. The findings were in agreement with the results from toxicity monitoring of shellfish by the Portuguese Institute for Sea and Atmosphere and confirm the suitability of the RNA microarray method for HABs detection and aquaculture management applications.     

Change in cyanobacterial biovolume due to preservation by Lugol's Iodine

Cyanobacterial biovolume is used as a guide to the public health risk from these organisms for users of potable and recreational waters. Most routine surveillance programs preserve phytoplankton samples before analysis. We tested the effect of Lugol's Iodine, a common preservative, on the cell biovolume of four common freshwater cyanobacteria, Microcystis aeruginosa, Anabaena circinalis, Cylindrospermopsis raciborskii and Aphanocapsa incerta. Linear dimensions and cell area were measured with an image analyser. All four species shrank after preservation. The magnitude of shrinkage varied with species and preservation time but was not affected by Lugol's concentration. The maximum shrinkage in each species was a 30–40% reduction compared to the live cell biovolume. These results suggest shrinkage can be a greater source of uncertainty in estimating the biovolume of toxigenic cyanobacteria in aquatic environments than natural variability in the cell dimensions, instrument precision or cell counting. Standardised cyanobacterial biovolume lists based on agreed geometric shapes and formulae would improve the value of this information for public health risk assessment.     

A shift in the dominant toxin-producing algal species in central California alters phycotoxins in food webs

In California, the toxic algal species of primary concern are the dinoflagellate Alexandrium catenella and members of the pennate diatom genus Pseudo-nitzschia, both producers of potent neurotoxins that are capable of sickening and killing marine life and humans. During the summer of 2004 in Monterey Bay, we observed a change in the taxonomic structure of the phytoplankton community—the typically diatom-dominated community shifted to a red tide, dinoflagellate-dominated community. Here we use a 6-year time series (2000–2006) to show how the abundance of the dominant harmful algal bloom (HAB) species in the Bay up to that point, Pseudo-nitzschia, significantly declined during the dinoflagellate-dominated interval, while two genera of toxic dinoflagellates, Alexandrium and Dinophysis, became the predominant toxin producers. This change represents a shift from a genus of toxin producers that typically dominates the community during a toxic bloom, to HAB taxa that are generally only minor components of the community in a toxic event. This change in the local HAB species was also reflected in the toxins present in higher trophic levels. Despite the small contribution of A. catenella to the overall phytoplankton community, the increase in the presence of this species in Monterey Bay was associated with an increase in the presence of paralytic shellfish poisoning (PSP) toxins in sentinel shellfish and clupeoid fish. This report provides the first evidence that PSP toxins are present in California's pelagic food web, as PSP toxins were detected in both northern anchovies (Engraulis mordax) and Pacific sardines (Sardinops sagax). Another interesting observation from our data is the co-occurrence of DA and PSP toxins in both planktivorous fish and sentinel shellfish. We also provide evidence, based on the statewide biotoxin monitoring program, that this increase in the frequency and abundance of PSP events related to A. catenella occurred not just in Monterey Bay, but also in other coastal regions of California. Our results demonstrate that changes in the taxonomic structure of the phytoplankton community influences the nature of the algal toxins that move through local food webs and also emphasizes the importance of monitoring for the full suite of toxic algae, rather than just one genus or species.     

Toxigenic phytoplankton and concomitant toxicity in the mussel Choromytilus meridionalis off the west coast of South Africa

The variability of toxigenic phytoplankton and the consequent uptake and loss of toxins by the mussel Choromytilus meridionalis was investigated in the southern Benguela at the event scale (3–10 days) in response to the upwelling–downwelling cycle. Phytoplankton and mussel samples were collected daily (20 March–11 April 2007) from a mooring station (32.04°S; 18.26°E) located 3.5 km offshore of Lambert's Bay, within the St Helena Bay region. Rapid changes in phytoplankton assemblages incorporated three groups of toxigenic phytoplankton: (1) the dinoflagellate Alexandrium catenella; (2) several species of Dinophysis, including Dinophysis acuminata, Dinophysis fortii, Dinophysis hastata and Dinophysis rotundata; and (3) members of the diatom genus Pseudo-nitzschia. Analysis of phytoplankton concentrates by LC–MS/MS or LC-FD provided information on the toxin composition and calculated toxicity of each group. Several additional in vitro assays were used for the analysis of toxins in mussels (ELISA, RBA, MBA for PSP toxins; and ELISA for DSP toxins). Good correspondence was observed between methods except for the MBA, which provided significantly lower (approximately 2-fold) estimates of PSP toxins. PSP and DSP toxins both exceeded the regulatory limits in Choromytilis meridionalis, but ASP toxins were undetected. Differences were observed in the composition of both PSP and DSP toxins in C. meridionalis from that of the ingested dinoflagellates (PSP toxins showed an increase in STX, C1,2, and traces of dcSTX and GTX1,4 and a decrease in NEO; DSP toxins showed an increased in DTX1, and traces of PTX2sa, and a decrease in OA). The rate of loss of PSP toxins following dispersal of the A. catenella boom was 0.12 d⁻¹. Variation in the loss rates of different PSP toxins contributed to the change in toxin profile in C. meridionalis. Prediction of net toxicity in shellfish of the nearshore environment in the southern Benguela is limited due to rapid phytoplankton community changes, high variability in cellular toxicity, and the selective uptake and loss of toxins, and/or transformation of toxins.     

Cylindrospermopsis raciborskii Virus and host: genomic characterization and ecological relevance

Cylindrospermopsis (Raphidiopsis) raciborskii is an invasive, filamentous, nitrogen-fixing cyanobacterium that forms frequent blooms in freshwater habitats. While viruses play key roles in regulating the abundance, production and diversity of their hosts in aquatic ecosystems, the role(s) of viruses in the ecology of C. raciborskii is almost unexplored. Progress in this field has been hindered by the absence of a characterized virus–host system in C. raciborskii. To bridge this gap, we sequenced the genome of CrV-01T, a previously isolated cyanosiphovirus, and its host, C. raciborskii strain Cr2010. Analyses suggest that CrV-01T represents a distinct clade of siphoviruses infecting, and perhaps lysogenizing, filamentous cyanobacteria. Its genome contains unique features that include an intact CRISPR array and a 12 kb inverted duplication. Evidence suggests CrV-01T recently gained the ability to infect Cr2010 and recently lost the ability to form lysogens. The cyanobacterial host contains a CRISPR-Cas system with CRISPR spacers matching protospacers within the inverted duplication of the CrV-01T genome. Examination of metagenomes demonstrates that viruses with high genetic identity to CrV-01T, but lacking the inverted duplication, are present in C. raciborskii blooms in Australia. The unique genomic features of the CrV/Cr2010 system offers opportunities to investigate in more detail virus–host interactions in an ecologically important bloom-forming cyanobacterium.