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.     

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.     

CYLINDROSPERMOPSIS RACIBORSKII (CYANOBACTERIA) INVASION AT MID‐LATITUDES: SELECTION,WIDE PHYSIOLOGICAL TOLERANCE,ORGLOBALWARMING?1

The tropical bloom‐forming cyanobacterium Cylindrospermopsis raciborskii (Woloszynska) Seenaya et Subba Raju is causing increasing concern because of its potential toxicity and invasive behavior at mid‐latitudes. This species has recently been identified in several temperate areas and was first reported in France in 1994, but the mechanisms underlying this acclimation process remain to be elucidated. We performed a range of laboratory experiments in an attempt to identify the physiological characteristics that could account for this behavior. We investigated the three following hypotheses: 1) clones of C. raciborskii adapted to temperate climate have been selected as it advanced north, 2) C. raciborskii has high physiological tolerance that allows it to prosper in a wide range of conditions, and 3) changes inclimate (global warming) have favored the development of C. raciborskii in mid‐latitudes. Ten strains of C. raciborskii from Australia n=1), Brazil (2), France (2), Germany (1), Hungary (1), Mexico (1), and Senegal (2) were cultured at different temperatures and light intensities. The in vitro growth parameters (μ and T_opt) were the same for the tropical and temperate strains. All 10 strains displayed positive net growth in a wide range of temperatures (from 20 to 35°C) and light intensities (from 30 to 400 μmol photons·m^?2·s^?1), with maximum growth rates at around 30°C and 80 μmol photons·m^?2·s^?1. This suggests that the colonization of mid‐latitudes by C. raciborskii may result from a combination of its ability to tolerate a rather wide range of climatic conditions and the global warming phenomenon, which provides this species with better environmental conditions for its growth.     

Growth and temperature‐related phenotypic plasticity in the cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is an invasive and potentially toxic cyanobacterium, which has recently spread worldwide, mainly because of its tolerance to a wide range of climatic conditions. C. raciborskii is able to change several traits in response to environmental changes and its morphology is also affected by these changes (especially in nutrients). We also expected temperature to affect the morphology of this cyanobacterium. We examined the growth and morphology of C. raciborskii at different temperatures and compared laboratory results to the morphology of this cyanobacterium in situ. As expected, growth rates increased with temperature. In addition, a high carrying capacity at 32°C suggests that this cyanobacterium is able to form more dense blooms at high temperatures. Fragile trichomes and low growth rates were observed at 12°C. An increase in the growth rate related to temperature resulted in a decrease in trichome length, with shorter trichomes at 32°C. The same pattern was observed in wild populations of C. raciborskii in a tropical reservoir, where shorter trichomes were observed in warmer months, when biomass was highest. This species' high ability to adapt to different environmental conditions throughout the year (i.e., nutrients, temperature) may have provided it with an additional advantage to increase its perennial blooms, mainly in tropical regions.     

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.     

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.     

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.     

Growth optimization of the invasive cyanobacterium Cylindrospermopsis raciborskii in response to phosphate fluctuations

The role of ecophysiological traits in the success and expansion of the toxic cyanobacterium Cylindrospermopsis raciborskii is still under debate. One key factor appears to be the high physiological flexibility of this organism when obtaining limiting resources. Recent studies have found that filamentous bloom-forming cyanobacteria are able to optimize their growth by adjusting phosphate uptake during fluctuating nutrient conditions. We investigated the growth response of two phosphate-deficient C. raciborskii isolates (MVCC19 from Uruguay and CCMP1973 from USA) to short-term fluctuations in phosphate supply. These isolates were exposed to five phosphate concentrations which were provided in two supply modes: a single pulse (SingleP) versus the same amount divided in 10 pulses (TenP), with one pulse applied every 6 min. Morphological traits and changes in chlorophyll a and phycocyanin fluorescence were also evaluated. Growth rates of CCMP1973 and MVCC19 almost doubled and tripled, respectively, when exposed to multiple rather than single phosphate additions. Different growth rates were observed with the same total added resource, thus contradicting the classical model of dependence of growth rate on absolute external concentration. Phosphate-deficient C. raciborskii showed a remarkable physiological flexibility in adapting to phosphate availability on a timescale from minutes to hours. The TenP mode provided an extension of phosphate exposure time that allowed the energetic optimization of uptake and growth. The morphological plasticity of the species in response to phosphate supply mode was also shown by differences in trichome length and individual size. Our results are the first evidence of growth optimization of phosphate-deficient C. raciborskii to short-term nutrient fluctuations, revealing its physiological flexibility. This adaptive behaviour may help to explain the invasive success of this diazotrophic cyanobacterium in a wide range of aquatic ecosystems where phosphorus is frequently the limiting resource.     

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.     

Evidence for differences in the metabolism of saxitoxin and C1+2 toxins in the freshwater cyanobacterium Cylindrospermopsis raciborskii T3

The activity of paralytic shellfish poisoning (PSP) toxins biosynthetic enzymes was assayed in the cyanobacterium Cylindrospermopsis raciborskii T3 after inhibiting protein synthesis with chloramphenicol (CAM). The production of C1+2 and saxitoxin (STX) was sensitive to CAM with STX levels decreasing by 70% after 24-h exposure to the antibiotic. PSP toxin production was strongly promoted by arginine supplementation, with a maximum 476% increase in intracellular STX concentrations after 24-h exposure to 10 mM of the amino acid. However, arginine had no stimulating effect on PSP toxin levels if supplemented in combination with CAM at 10 microg l(-1). Addition of agmatine and proline to C. raciborskii T3 cultures in the presence of 10 microg l(-1) CAM increased C1+2 toxins levels, while having a negative or no effect on STX accumulation. In vitro, PSP toxin levels increased naturally in cyanobacterial extracts, with CAM and arginine having no influence on either C1+2 or STX synthesis. The evidence presented in this study suggests a possible difference between the metabolism of STX and the C1+2 toxins and indicated a high turnover rate of STX biosynthetic enzymes in C. raciborskii T3.     

Effects of Saxitoxin (STX) and Veratridine on Bacterial Na+-K+ Fluxes: a Prokaryote-Based STX Bioassay

Saxitoxin (STX) is a potent natural sodium channel blocker and represents a significant health concern worldwide. We describe here the antagonistic effects of STX and veratridine (VTD), an Na⁺ channel activator, on three gram-negative bacteria and their application to an STX bioassay. STX reduced the total cellular levels of both Na⁺ and K⁺, as measured by flame photometry, whereas VTD increased the cellular concentrations relative to control ion fluxes in the cyanobacterium Cylindrospermopsis raciborskii AWT205. Endogenous STX production in toxic cyanobacterial strains of C. raciborskii and Anabaena circinalis prevented cell lysis induced by VTD stress. Microscopic cell counts showed that non-STX producing cyanobacteria displayed complete cell lysis and trichome fragmentation 5 to 8 h after addition of VTD and vanadate (VAN), an inhibitor of sodium pumps. The addition of STX, or its analogue neoSTX, prior to treatment with VTD plus VAN prevented complete lysis in non-STX-producing cyanobacteria. VTD also affected cyanobacterial metabolism, and the presence of exogenous STX in the sample also ameliorated this decrease in metabolic activity, as measured by the cellular conversion of tetrazolium into formazan. Reduced primary metabolism was also recorded as a decrease in the light emissions of Vibrio fischeri exposed to VTD. Addition of STX prior to VTD resulted in a rapid and dose-dependent response to the presence of the channel blocker, with samples exhibiting resistance to the VTD effect. Our findings demonstrate that STX and VTD influence bacterial Na⁺ and K⁺ fluxes in opposite ways, and these principles can be applied to the development of a prokaryote-based STX bioassay.     

Zooplankton community structure, micro-zooplankton grazing impact, and seston energy content in the St. Johns river system, Florida as influenced by the toxic cyanobacterium Cylindrospermopsis raciborskii

Zooplankton can influence the phytoplankton community through preferential grazing. In turn, nuisance cyanobacteria may affect zooplankton community structure by allowing certain species to out-compete others. We examined zooplankton-phytoplankton interactions, micro-zooplankton (< 200 m) grazing, and biochemical components of the seston in the St. Johns River System (SJR), Florida in the presence and absence of the toxin-producing cyanobacterium Cylindrospermopsis raciborskii. We tested whether this cyanobacterium would cause a decrease in the size structure of the zooplankton community and postulated a resultant decline in the metabolic energy and carbon available to higher consumers (i.e. fish). When numbers of C. raciborskii were low or undetectable, zooplankton were more diverse and were comprised of larger species. Rotifers were the dominant zooplankton, and their numbers relative to other zooplankton increased as C. raciborskii concentrations increased. Micro-zooplankton grazing was higher in times of C. raciborskii abundance, suggesting competitive and predatory exclusion by larger zooplankton in times of higher phytoplankton diversity. Total caloric content of the seston was higher in times of C. raciborskii abundance. However, essential fatty acids and phosphorus may be lacking in the seston, or nutrients may potentially be sequestered by the cyanobacteria and remain as organic matter in the water column. In such cases, higher trophic levels would not be able to obtain optimal energy requirements. Overall, there was a greater impact of micro-grazers on phytoplankton in the presence of C. raciborskii and apparent negative effects on the larger zooplankton species, suggesting a potential for changes in zooplankton and higher trophic level community structure.     

Growth and phosphate uptake kinetics of the cyanobacterium,Cylindrospermopsis raciborskii (Cyanophyceae) in throughflow cultures

• 1 Cylindrospermopsis raciborskii occupies a rapidly expanding geographical area. Its invasive success challenges eutrophication control in many lakes. To understand better the load‐dependent behaviour of this nitrogen fixing cyanobacterium under in situ conditions, we studied P‐dependent growth of a C. raciborskii strain under continuous and pulsed P supply.
• 2 The Droop model reasonably described P‐dependent growth in the continuously supplied chemostats. Large P pulses, however, caused a delay in growth and cells subject to P pulses grew slower than their counterparts with the same P quota supplied continuously.
• 3 The kinetics of P uptake indicated that C. raciborskii is opportunistic with respect to P. Its high excess P storage capacity after a saturating P pulse (Q_ex=95 µg P [mg C]^‐1) and P‐specific uptake capacity (U_max = V_max/Q_P=150–1200) are indicative of storage adaptation. At the same time, the affinity of the P uptake system (U_max/K = 800–4000) is also high.
• 4 Rate of leakage exceeded that of the steady state net P uptake by one to two orders of magnitude. Growth affinity of C. raciborskii (µ_max/K_µ≈ 20) was relatively low, presumably due to the substantial leakage.
• 5 The dynamics of the particular water body determine which trait contributes most to competitive success of C. raciborskii. In deep lakes with vertical nutrient gradients, the cyanobacterium may rely primarily on its high P storage capacity, which is coupled to a lack of short‐term feedback inhibition and efficient buoyancy regulation. In lakes without such gradients, high P uptake affinity may be vitally important.

     

Effects of iron on growth and reflectance spectrum of the bloom‐forming cyanobacterium Microcystis viridis

Iron is an important factor in algal blooms because it is involved in cyanobacterial pigment biosynthesis and therefore has the ability to influence the pigment status of algal cells. This role in pigment biosynthesis offers the opportunity for rapid monitoring of iron availability to cyanobacteria through spectral reflectance characterization. In the present study, the freshwater cyanobacterium Microcystis viridis was cultured with different levels of iron. Cell density, cellular content of iron and photosynthetic pigments, and spectral reflectivity of M. viridis were determined daily during the course of the culture experiment. The results showed that at the lowest iron concentration (0.01 μM) the growth of M. viridis was seriously limited, and the maximal cell density was only approximately 6.4% of the density observed with an iron concentration of 18 μM. Iron availability dramatically affected chlorophyll a, carotenoid and phycocyanin content, with the greatest impact on chlorophyll a. The iron‐induced changes in content and ratios of pigments were detectable through spectral reflectance. Eleven spectral indices previously developed for the estimation of concentrations and/or ratios of pigments and a newly proposed chlorophyll a/phycocyanin index were found to be suitable for generating sensitive regression models between cellular iron content and spectral parameters. The comprehensive application of key sensitive spectral indices and regression equations should help to support monitoring and diagnosis of iron availability to cyanobacteria via remote sensing.     

BIOCHEMICAL CHARACTERIZATION OF PARALYTIC SHELLFISH TOXIN BIOSYNTHESIS IN VITRO1

Saxitoxin (STX) and its analogs are voltage‐gated sodium‐channel blockers that cause paralytic shellfish poisoning (PSP) and negatively affect human health and seafood industries worldwide. Little is known about the molecular biology of PSP‐toxin synthesis. Saxitoxin precursors were identified 25 years ago, and a hypothetical biosynthesis pathway was proposed; however, the correct sequence of reactions and enzymes involved in their catalysis remains to be identified. This study describes the optimization of in vitro biosynthesis of PSP toxins by cellular lysates of the toxic cyanobacterium Cylindrospermopsis raciborskii (Wo?osz.) Seenaya et Subbaraju T3 and the characterization of its biochemical requirements. Enzymes involved in PSP‐toxin synthesis are located in the cytosol. The molecular components of in vitro biosynthesis reactions could not be completely defined because of the requirement of an unknown cofactor. Evidence is presented that supports the previous suggestion that STX biosynthesis involves a Claisen condensation between arginine and acetate. In addition, carbamoyl phosphate was identified as a likely precursor for carbamated PSP toxins. Predictions have been made regarding the enzymes that may be involved in the biosynthesis of PSP toxins. These included class II aminotransferase; nonheme iron oxygenase, containing flavin, and possibly ferredoxin, as the prosthetic groups; and an O‐carbamoyltransferase. On the other hand, the involvement of cytochrome P450 monooxygenase was excluded.     

Syntaxin 3 SPI-2 dependent crosstalk facilitates the division of Salmonella containing vacuole

Intracellular membrane fusion is mediated by membrane-bridging complexes of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE proteins are one of the key players in vesicular transport. Several reports shed light on intracellular bacteria modulating host SNARE machinery to establish infection successfully. The critical SNAREs in macrophages responsible for phagosome maturation are Syntaxin 3 (STX3) and Syntaxin 4 (STX4). Reports also suggest that Salmonella actively modulates its vacuole membrane composition to escape lysosomal fusion. Salmonella containing vacuole (SCV) harbours recycling endosomal SNARE Syntaxin 12 (STX12). However, the role of host SNAREs in SCV biogenesis and pathogenesis remains unclear. Upon knockdown of STX3, we observed a reduction in bacterial proliferation, which is concomitantly restored upon the overexpression of STX3. Live-cell imaging of Salmonella-infected cells showed that STX3 localises to the SCV membranes and thus might help in the fusion of SCV with intracellular vesicles to acquire membrane for its division. We also found the interaction STX3-SCV was abrogated when we infected with SPI-2 encoded Type 3 secretion system (T3SS) apparatus mutant (STM ∆ssaV) but not with SPI-1 encoded T3SS apparatus mutant (STM ∆invC). These observations were also consistent in the mice model of Salmonella infection. Together, these results shed light on the effector molecules secreted through T3SS encoded by SPI-2, possibly involved in interaction with host SNARE STX3, which is essential to maintain the division of Salmonella in SCV and help to maintain a single bacterium per vacuole.     

Evidence of the existence of a toxic form of Cylindrospermopsis raciborskii (Nostocales,Cyanobacteria) in Japan

Cylindrospermopsis raciborskii is a common, bloom‐forming, planktonic, freshwater cyanobacterium. Toxic populations producing cylindrospermopsin can cause water‐safety problems. Although C. raciborskii is distributed worldwide, the presence of cylindrospermopsin‐producing strains of C. raciborskii was initially reported only in Australia and recently in Thailand. Here, we report the isolation of a toxic strain of C. raciborskii (ISG9) from a freshwater sample collected in Okinawa in 2008. This is the first report describing toxin expression in this species in Japan, detected from a subtropical area. The C. raciborskii species is known to produce cylindrospermopsin as a dominant toxin; however, in this new isolate, the dominant toxin expressed was deoxy‐cylindrospermopsin. The discovery of a toxic strain of C. raciborskii in southern Japan emphasizes the need for basic monitoring schemes for this species in water supplies located in the temperate regions of Japan because of its possible expansion and distribution to other geographic areas.     

Effects of saxitoxin- and non-saxitoxin-producing strains of the cyanobacterium Cylindrospermopsis raciborskii on the fitness of temperate and tropical cladocerans

Life-table experiments were performed with two strains of Cylindrospermopsis raciborskii, a saxitoxin-producing strain (T3) and a non-saxitoxin-producing strain (NPLP-1) combined with the green algae Ankistrodesmus falcatus, aiming to detect effects on the reproduction of three cladocerans species: Daphnia pulex, Daphnia gessneri and Moina micrura. Survivorship, age at first reproduction, clutch size and population growth rate during 12–15 days were recorded. Cladocerans showed different susceptibility to saxitoxin-producing strain (T3), with D. pulex and M. micrura being negatively affected, followed by and D. gessneri, which did not show any decrease in fitness and performed even better than the controls with this strain. A reverse response was found in experiments with the non-saxitoxin-producing strain (NPLP-1): while D. pulex and M. micrura had an increase, D. gessneri showed a significant decrease in fitness, suggesting that this strain may produce some bioactive compound. The contrasting responses of the cladoceran species to saxitoxins and non-saxitoxin-producing strains of C. raciborskii suggest that the presence of this cyanobacterium may be a selective factor in determining the composition of zooplankton communities.     

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.     

PHOSPHORUS AND THE GROWTH OF JUVENILE MACROCYSTIS PYRIFERA (PHAEOPHYTA) SPOROPHYTES1

The effect of phosphate (P_i) supply on growth rate and tissue phosphorus content of juvenile Macrocystis pyrifera (L.) C. Ag. sporophytes was examined. Sporophytes were batch cultured in aquaria with flowing recirculated seawater enriched by 30 μM nitrate. Each aquarium was supplemented with a different seawater P_i concentration, 0, 0.3, 1, 2, 3, and 6 μM. Sporophyte mean specific growth rates declined with time in all cultures presumably due to the normal developmental decrease in the proportion of meristematic tissue of each plant. Growth rate declines were more pronounced in cultures that were nutrient limited. Sporophyte growth was P-limited after two-week exposure to P_i less than 1 μM, corresponding to a tissue P concentration of less than 0.20% dry weight. Plants cultured at 6 μM P_i contained tissue P levels of 0.53% dry weight after three weeks. Luxury consumption and storage of P occurred.