Anusuya Willis

Understanding the winning strategies used by the bloom-forming cyanobacterium Cylindrospermopsis raciborskii

The cyanobacterium Cylindrospermopsis raciborskii is a widespread species increasingly being recorded in freshwater systems around the world. It is of particular concern because strains in some geographic areas are capable of producing toxins with implications for human and animal health. Studies of this species have increased rapidly in the last two decades, especially in the southern hemisphere where toxic strains are prevalent. A clearer picture is emerging of the strategies adopted by this species to bloom and out-compete other species. This species has a high level of flexibility with respect to light and nutrients, with higher temperatures and carbon dioxide also promoting growth. There are two types of toxins produced by C. raciborskii: cylindrospermopsins (CYNs) and saxitoxins (STXs). The toxins CYNs are constitutively produced irrespective of environmental conditions and the ecological or physiological role is unclear, while STXs appear to serve as protection against high salinity and/or water hardness. It is also apparent that strains of this species can vary substantially in their physiological responses to environmental conditions, including CYNs production, and this may explain discrepancies in findings from studies in different geographical areas. The combination of a flexible strategy with respect to environmental conditions, and variability in strain response makes it a challenging species to manage. Our ability to improve bloom prediction will rely on a more detailed understanding of the complex physiology of this species.

Nutrient-related changes in the toxicity of field blooms of the cyanobacterium, Cylindrospermopsis raciborskii.

Nutrients have the capacity to change cyanobacterial toxin loads via growth-related toxin production, or shifts in the dominance of toxic and nontoxic strains. This study examined the effect of nitrogen (N) and phosphorus on cell division and strain-related changes in production of the toxins, cylindrospermopsins (CYNs) by the cyanobacterium, Cylindrospermopsis raciborskii. Two short-term experiments were conducted with mixed phytoplankton populations dominated by C. raciborskii in a subtropical reservoir where treatments had nitrate (NO3 ), urea (U) and inorganic phosphorus (P) added alone or in combination. Cell division rates of C. raciborskii were only statistically higher than the control on day 5 when U and P were co-supplied. In contrast, cell quotas of CYNs (QCYNS ) increased significantly in treatments where P was supplied, irrespective of whether N was supplied, and this increase was not necessarily related to cell division rates. Increased QCYNS did correlate with an increase in the proportion of the cyrA toxin gene to 16S genes in the C. raciborskii-dominated cyanobacterial population. Therefore, changes in strain dominance are the most likely factor driving differences in toxin production between treatments. Our study has demonstrated differential effects of nutrients on cell division and strain dominance reflecting a C. raciborskii population with a range of strategies in response to environmental conditions.

Intraspecific variation in growth, morphology and toxin quotas for the cyanobacterium, Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a bloom forming cyanobacterium with complex population dynamics and toxicity. In January of 2013 a single sample was collected from surface waters in Lake Wivenhoe, Australia, and twenty-four individual trichomes were isolated. Each isolate exhibited differences in growth rate, toxin cell quota and morphology, in the absence of phylogenetic heterogeneity. This study demonstrates substantial intraspecific isolate variation within a small sample and this has implications for understanding the population dynamics of this species.

Constitutive Cylindrospermopsin Pool Size in Cylindrospermopsis raciborskii under Different Light and CO2 Partial Pressure Conditions

ABSTRACT Cylindrospermopsin (CYN) and 7-deoxy-cylindrospermopsin (dCYN) are potent hepatotoxic alkaloids produced by numerous species of cyanobacteria, including the freshwater Cylindrospermopsis raciborskii. C. raciborskii is an invasive cyanobacterium, and the study of how environmental parameters drive CYN production has received significant interest from water managers and health authorities. Light and CO2 affect cell growth and physiology in photoautotrophs, and these are potential regulators of cyanotoxin biosynthesis. In this study, we investigated how light and CO2 affect CYN and dCYN pool size as well as the expression of the key genes, cyrA and cyrK, involved in CYN biosynthesis in a toxic C. raciborskii strain. For cells growing at different light intensities (10 and 100 μmol photons m−2 s−1), we observed that the rate of CYN pool size production (μCYN) was coupled to the cell division rate (μc) during batch culture. This indicated that CYN pool size under our experimental conditions is constant and cell quotas of CYN (QCYN) and dCYN (QdCYN) are fixed. Moreover, a lack of correlation between expression of cyrA and total CYN cell quotas (QCYNs) suggests that the CYN biosynthesis is regulated posttranscriptionally. Under elevated CO2 (1,300 ppm), we observed minor effects on QCYN and no effects on expression of cyrA and cyrK. We conclude that the CYN pool size is constitutive and not affected by light and CO2 conditions. Thus, C. raciborskii bloom toxicity is determined by the absolute abundance of C. raciborskii cells within the water column and the relative abundance of toxic and nontoxic strains.

Nitrogen fixation by the diazotroph Cylindrospermopsis raciborskii (Cyanophyceae).

Nitrogen fixation has been proposed as a mechanism that allows the diazotrophic cyanobacterium, Cylindrospermopsis raciborskii, to bloom in nitrogen‐limited freshwater systems. However, it is unclear whether dinitrogen fixation (N2 fixation) can supplement available dissolved inorganic nitrogen (DIN) for growth, or only provides minimum nitrogen (N) for cell maintenance under DIN deplete conditions. Additionally, the rate at which cells can switch between DIN use and N2 fixation is unknown. This study investigated N2 fixation under a range of nitrate concentrations. Cultures were grown with pretreatments of nitrate replete (single dose 941 μmol NO3− · L−1) and N‐free conditions and then either received a single dose of 941 μmol NO3− · L−1 (N941), 118 μmol NO3− · L−1 (N118) or 0 N. Heterocysts appeared from days 3 to 5 when treatments of high NO3− were transferred to N free media (N941:N0), and from day 5 in N941 transferred to N118 treatments. Conversely, transferring cells from N0 to N941 resulted in heterocysts being discarded from day 3 and day 5 for N0:N118. Heterocyst appearance correlated with a detectable rate of N2 fixation and up‐regulation of nifH gene expression, the discard of heterocysts occurred after sequential reduction of nifH expression and N2 fixation. Nitrate uptake rates were not affected by pretreatment, suggesting no regulation or saturation of this uptake pathway. These data demonstrate that for C. raciborskii, N2 fixation is regulated by the production or discard of heterocysts. In conclusion, this study has shown that N2 fixation only provides enough N to support relatively low growth under N‐limited conditions, and does not supplement available nitrate to increase growth rates.

Nitrogen fixation by the reluctant diazotroph Cylindrospermopsis raciborskii (Cyanophyceae)

Nitrogen fixation has been proposed as a mechanism that allows the diazotrophic cyanobacterium, Cylindrospermopsis raciborskii, to bloom in nitrogen‐limited freshwater systems. However, it is unclear whether dinitrogen fixation (N2 fixation) can supplement available dissolved inorganic nitrogen (DIN) for growth, or only provides minimum nitrogen (N) for cell maintenance under DIN deplete conditions. Additionally, the rate at which cells can switch between DIN use and N2 fixation is unknown. This study investigated N2 fixation under a range of nitrate concentrations. Cultures were grown with pretreatments of nitrate replete (single dose 941 μmol NO3− · L−1) and N‐free conditions and then either received a single dose of 941 μmol NO3− · L−1 (N941), 118 μmol NO3− · L−1 (N118) or 0 N. Heterocysts appeared from days 3 to 5 when treatments of high NO3− were transferred to N free media (N941:N0), and from day 5 in N941 transferred to N118 treatments. Conversely, transferring cells from N0 to N941 resulted in heterocysts being discarded from day 3 and day 5 for N0:N118. Heterocyst appearance correlated with a detectable rate of N2 fixation and up‐regulation of nifH gene expression, the discard of heterocysts occurred after sequential reduction of nifH expression and N2 fixation. Nitrate uptake rates were not affected by pretreatment, suggesting no regulation or saturation of this uptake pathway. These data demonstrate that for C. raciborskii, N2 fixation is regulated by the production or discard of heterocysts. In conclusion, this study has shown that N2 fixation only provides enough N to support relatively low growth under N‐limited conditions, and does not supplement available nitrate to increase growth rates.

Differences in cyanobacterial strain responses to light and temperature reflect species plasticity

Microcystis aeruginosa and Cylindrospermopsis raciborskii are two cyanobacterial species that dominate freshwaters globally. Multiple strains of each species with different physiology occur, however, many studies have focused only on one or two strains, limiting our understanding of both strain variation and characterisation of the species. Therefore, in this study we examined the variation in growth and morphology of multiple isolates of both species, isolated from two adjacent Australian reservoirs. Four M. aeruginosa strains (=isolates) (one colony-forming, three single-celled morphology) and eight C. raciborskii isolates (five with straight trichomes, three with coiled trichomes) were cultured individually in a factorial designed experiment with four light intensities (L: 10, 30, 50 and 100μmol photons m-2s-1) and two temperatures (T: 20 and 28°C). The specific growth rate (μ), cell volume, and final cell concentration was measured. The light attenuation coefficient (kj), a measure of self-shading, was calculated. The results showed that the intraspecific variation was greater than the interspecific variation. The μ of all isolates of M. aeruginosa and C. raciborskii ranged from 0.16 to 0.55d-1 and 0.15 to 0.70d-1, respectively. However, at a specific light and temperature the mean μ of all M. aeruginosa isolates and C. raciborskii isolates were similar. At the species level, M. aeruginosa had higher growth rates at higher light intensity but lower temperature (L100T20), while straight C. raciborskii had higher growth rates at lower light intensity but higher temperature (L50T28), and coiled C. raciborskii had higher growth rates at higher light intensity and higher temperature (L100T28). The final cell concentrations of M. aeruginosa were higher than C. raciborskii. However, C. raciborskii isolates had greater variation in μ, kj and cell volume than M. aeruginosa. kj varied with light and temperature, and decreased with surface-to-volume ratio within each species. kj was lower for M. aeruginosa compared to C. raciborskii as expected based on cell size, but interestingly, C. raciborskii coiled isolates had lower kj than the straight isolates suggesting lower effect of self-shading. This study highlights the extent of strain variation to environmental conditions and to species variability.

Review: a meta-analysis comparing cell-division and cell-adhesion in Microcystis colony formation

The freshwater cyanobacterium Microcystis is a nuisance species. It forms large blooms on the water surface and overwhelmingly dominates the ecosystem through the formation of colonies from single cells surrounded by mucilage; however, the mechanism of colony formation is poorly understood. Two mechanisms of Microcystis colony formation have been proposed: cell-division, where cells remain attached after binary fission; and cell-adhesion, where single cells stick together. This paper examined the published literature on Microcystis colony formation to clarify the mechanism of colony formation and its relationship to environmental drivers. This meta-analysis showed that in laboratory experiments, colony formation by cell-division was mainly induced by zooplankton filtrate, high Pb2+ concentrations, the presence of the cyanobacterium Cylindrospermopsis raciborskii, heterotrophic bacteria, and low temperature and low light intensities. Alternatively, colony formation by cell-adhesion was mainly induced by zooplankton grazing, high Ca2+ concentrations, and microcystins. Therefore, colony formation by cell-division appears to be a slower process and to occur under an environmental stress factor, while cell-adhesion occurs more quickly to an environmental threat. Applying the criteria to the different morphospecies of Microcystis, it was found that under natural conditions M. ichthyoblabe colonies formed predominantly through cell-division, whereas M. wesenbergii colonies formed predominantly through cell-adhesion. This study provides new insights into the mechanisms and environmental drivers of colony formation by Microcystis.

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

Abstract 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 (Ks = 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.

Application of first order rate kinetics to explain changes in bloom toxicity-the importance of understanding cell toxin quotas

Cyanobacteria are oxygenic photosynthetic Gram-negative bacteria that can form potentially toxic blooms in eutrophic and slow flowing aquatic ecosystems. Bloom toxicity varies spatially and temporally, but understanding the mechanisms that drive these changes remains largely a mystery. Changes in bloom toxicity may result from changes in intracellular toxin pool sizes of cyanotoxins with differing molecular toxicities, and/or from changes in the cell concentrations of toxic and non-toxic cyanobacterial species or strains within bloom populations. We show here how first-order rate kinetics at the cellular level can be used to explain how environmental conditions drive changes in bloom toxicity at the ecological level. First order rate constants can be calculated for changes in cell concentration (μc: specific cell division rate) or the volumetric biomass concentration (μg: specific growth rate) between short time intervals throughout the cell cycle. Similar first order rate constants can be calculated for changes in nett volumetric cyanotoxin concentration (μtox: specific cyanotoxin production rate) over similar time intervals. How μc (or μg) covaries with μtox over the cell cycle shows conclusively when cyanotoxins are being produced and metabolised, and how the toxicity of cells change in response to environment stressors. When μtox/μc >1, cyanotoxin cell quotas increase and individual cells become more toxic because the nett cyanotoxin production rate is higher than the cell division rate. When μtox / μc =1, cell cyanotoxin quotas remains fixed because the nett cyanotoxin production rate matches the cell division rate. When μtox/μc <1, the cyanotoxin cell quota decreases because either the nett cyanotoxin production rate is lower than the cell division rate, or metabolic breakdown and/ or secretion of cyanotoxins is occurring. These fundamental equations describe cyanotoxin metabolism dynamics at the cellular level and provide the necessary physiological background to understand how environmental stressors drive changes in bloom toxicity.