Enhanced resistance of co-existing toxigenic and non-toxigenic Microcystis aeruginosa to pyrogallol compared with monostrains.

Cyanobacteria species are sensitive to many plant allelochemicals, such as pyrogallol. However, little attention has been paid to the relative effects of these xenobiotics on co-occurring toxigenic and non-toxigenic cyanobacterial strains, despite their co-existence in blooms. Hence, the responses of one toxigenic (TS2) and two non-toxigenic (NS1, NS2) Microcystis aeruginosa strains to pyrogallol were tested under three conditions: mono-culture and co-cultured either directly or separately by dialysis membrane. The study showed that the inhibitory effects of pyrogallol on the growth and photosynthetic yield (Fv/Fm) of either toxigenic or non-toxigenic M. aeruginosa strains were lower in direct and dialysis co-culture conditions than those in mono-culture conditions. This result indicated that chemical-mediated reciprocal effects occur between the co-existing toxigenic and non-toxigenic strains. The toxigenic M. aeruginosa strain was more sensitive to pyrogallol than the non-toxigenic strains in both mono- and co-culture systems, though whether this outcome is due to the former's toxigenic status is unclear. Intracellular microcystin-LR (MC-LR) concentrations of the toxigenic strain decreased after pyrogallol addition in both mono- and co-culture systems, whereas extracellular MC-LR concentrations increased. This finding may reflect the cell damage of M. aeruginosa because of the pyrogallol. At the same initial number of cells, the extracellular MC-LR concentration released from the same amount of TS2 cells in mono-culture was slightly higher than that in dialysis co-culture conditions. Overall, this study shows that plant allelochemicals may have the potential to reduce bloom toxicity by reducing the proportion of toxigenic cyanobacterial strains, and the effects of co-existing strains must be considered when assessing the effects of plant allelochemicals on target strains.

Subtropical freshwater phytoplankton show a greater response to increased temperature than to increased pCO2.

Global increases in atmospheric CO2 and temperatures will impact aquatic systems, with freshwater habitats being affected. Some studies suggest that these conditions will promote cyanobacterial dominance. There is a need for a clearer picture of how algal species and strains within species will respond to higher temperatures and CO2, especially in combination. This study examined two chlorophytes (Monoraphidium and Staurastrum), and two strains of the cyanobacterium Raphidiopsis raciborskii (straight S07 and coiled C03), to determine how the combination of higher temperature and CO2 levels will affect their growth and maximum cell concentrations. Continuous cultures were used to compare the steady state cell concentrations at 28 °C and 30 °C, and CO2 partial pressures (pCO2), 400 and 750 ppm for all cultures, and in addition 1000 ppm at 28 °C for R. raciborskii strains. This study showed that, for all species, water temperature had a greater effect than higher pCO2 on cell concentrations. There were clear differences in response between the chlorophyte species, with Monoraphidium preferring 28 °C and Staurastrum preferring 30 °C. There were also differences in response of the R. raciborskii strains to increasing temperature and pCO2, with S07 having a greater increase in cell concentration. Genome analysis of R. raciborskii showed that the straight strain has five additional carbon acquisition genes (ß-CA, chpY, cmpB, cmpD and NdhD4), indicative of increased carbon metabolism. These differences in the strains' response to elevated pCO2 will lead to changes in the species population structure and distribution in the water column. This study shows that it is important to examine the effects of both pCO2 and temperature, and to consider strain variation, to understand how species composition of natural systems may change under future climate conditions.

Causal relationships of Raphidiopsis (formerly Cylindrospermopsis) dynamics with water temperature and N:P-ratios: A meta-analysis across lakes with different climates based on inferential modelling.

Raphidiopsis raciborskii is a tropical toxic cyanobacterium that is rapidly expanding to diverse lake habitats in different climate zones by sophisticated adaptation mechanisms. This meta-analysis investigated correlations of R. raciborskii with water temperature and N:P (nitrogen to phosphorus)-ratios across four lakes with different climates and trophic states by means of long-term time series and the hybrid evolutionary algorithm HEA. The results have shown that in the lakes with temperate and Mediterranean climate, R. raciborskii is strongly correlated with water temperature since germination and growth rely on rising water temperatures in spring. In contrast, there was a weaker correlation with water temperature in subtropical and tropical lakes where pelagic populations of R. raciborskii are overwintering, and are present all year round. However, the highest abundances of R. raciborskii coincided with highest water temperature for the Mediterranean, subtropical and tropical lakes, whilst in the temperate Langer See the highest abundances of R. raciborskii occurred at 24.1 °C, even though temperatures of up to 27 °C were recorded in 2013 and 2014. The correlation of R. raciborskii with N:P-ratios proved to be strongest for the meso- to eutrophic Lake Kinneret (r2 = 0.8) and lowest for the eutrophic Lake Paranoa (r2 = 0.16). However, the assumption has been confirmed that R. raciborskii is growing fastest when waters are N-limited regardless of trophic states. In terms of phenology, the temperate and Mediterranean lakes displayed "fastest growth" in spring and early summer. In contrast, the growing season in subtropical and tropical lakes lasted from spring to autumn most likely because of overwintering populations, and growing importance of direct and indirect biotic regulating factors such as competition, grazing, remineralisation of nutrients along warming climate. In order to carry out a meta-analysis of time series across four different lakes, HEA served as powerful tool resulting in inferential models with predictive capacity for population dynamics of R. raciborskii just driven by water temperature or N:P-ratios, whilst coefficients of determination r2 served as criteria for hypotheses testing.

Early warning of limit-exceeding concentrations of cyanobacteria and cyanotoxins in drinking water reservoirs by inferential modelling.

An early warning scheme is proposed that runs ensembles of inferential models for predicting the cyanobacterial population dynamics and cyanotoxin concentrations in drinking water reservoirs on a diel basis driven by in situ sonde water quality data. When the 10- to 30-day-ahead predicted concentrations of cyanobacteria cells or cyanotoxins exceed pre-defined limit values, an early warning automatically activates an action plan considering in-lake control, e.g. intermittent mixing and ad hoc water treatment in water works, respectively. Case studies of the sub-tropical Lake Wivenhoe (Australia) and the Mediterranean Vaal Reservoir (South Africa) demonstrate that ensembles of inferential models developed by the hybrid evolutionary algorithm HEA are capable of up to 30days forecasts of cyanobacteria and cyanotoxins using data collected in situ. The resulting models for Dolicospermum circinale displayed validity for up to 10days ahead, whilst concentrations of Cylindrospermopsis raciborskii and microcystins were successfully predicted up to 30days ahead. Implementing the proposed scheme for drinking water reservoirs enhances current water quality monitoring practices by solely utilising in situ monitoring data, in addition to cyanobacteria and cyanotoxin measurements. Access to routinely measured cyanotoxin data allows for development of models that predict explicitly cyanotoxin concentrations that avoid to inadvertently model and predict non-toxic cyanobacterial strains.

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.

Constitutive cylindrospermopsin pool size in Cylindrospermopsis raciborskii under different light and CO2 partial pressure conditions.

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.

Elevated CO2 causes changes in the photosynthetic apparatus of a toxic cyanobacterium, Cylindrospermopsis raciborskii.

We studied the physiological acclimation of growth, photosynthesis and CO2-concentrating mechanism (CCM) in Cylindrospermopsis raciborskii exposed to low (present day; L-CO2) and high (1300ppm; H-CO2) pCO2. Results showed that under H-CO2 the cell specific division rate (µc) was higher and the CO2- and light-saturated photosynthetic rates (Vmax and Pmax) doubled. The cells' photosynthetic affinity for CO2 (K0.5CO2) was halved compared to L-CO2 cultures. However, no significant differences were found in dark respiration rates (Rd), pigment composition and light harvesting efficiency (α). In H-CO2 cells, non-photochemical quenching (NPQ), associated with state transitions of the electron transport chain (ETC), was negligible. Simultaneously, a reorganisation of PSII features including antenna connectivity (JconPSIIα), heterogeneity (PSIIα/ß) and effective absorption cross sectional area (σPSIIα/ß) was observed. In relation to different activities of the CCM, our findings suggest that for cells grown under H-CO2: (1) there is down-regulation of CCM activity; (2) the ability of cells to use the harvested light energy is altered; (3) the occurrence of state transitions is likely to be associated with changes of electron flow (cyclic vs linear) through the ETC; (4) changes in PSII characteristics are important in regulating state transitions.

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.

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.

Investigating the production and release of cylindrospermopsin and deoxy-cylindrospermopsin by Cylindrospermopsis raciborskii over a natural growth cycle.

Many harmful cyanobacterial genera have strains that can produce potent toxins and other biologically active compounds that present a risk to the health of humans and other animals that consume or contact contaminated water. Cylindrospermopsins (CYNs) are produced by several species of cyanobacteria including Cylindrospermopsis raciborskii (Woloszynska) Seenayya and Subba Raju. Previous studies have used filtration methods to separate between the particulate and dissolved CYNs pools. Filtration may lyse cells and thus overestimate the dissolved CYNs pool. Here we employed a novel passive sampling technique to measure the proportion of dissolved CYNs in two Australian strains of C. raciborskii over the growth cycle while minimizing potential overestimation of the dissolved CYNs pool. We simultaneously compared the ratios of the two major CYNs produced by Australian strains of C. raciborskii: cylindrospermopsin (CYN) and deoxy-CYN in the particulate and dissolved pools. CYNs stayed within the cells during log phase but accumulated in the water column during stationary and senescent phases. The proportion of deoxy-CYN to CYN differed between strains but increased in both as cells aged. We conclude that while active release or leaking of CYNs from actively growing cells does occur, CYNs in the water column were primarily a result of cell lysis during stationary phase or due to other environmental stressors. The production of CYN and deoxy-CYN were a constitutive process and both the concentration of, ratio between, and release of CYN and deoxy-CYN were strain dependent. Future studies must account for the genetic diversity of CYN producers when investigating the production of CYNs in natural systems.

Inductive reasoning and forecasting of population dynamics of Cylindrospermopsis raciborskii in three sub-tropical reservoirs by evolutionary computation.

Seven-day-ahead forecasting models of Cylindrospermopsis raciborskii in three warm-monomictic and mesotrophic reservoirs in south-east Queensland have been developed by means of water quality data from 1999 to 2010 and the hybrid evolutionary algorithm HEA. Resulting models using all measured variables as inputs as well as models using electronically measurable variables only as inputs forecasted accurately timing of overgrowth of C. raciborskii and matched well high and low magnitudes of observed bloom events with 0.45≤r2>0.61 and 0.4≤r2>0.57, respectively. The models also revealed relationships and thresholds triggering bloom events that provide valuable information on synergism between water quality conditions and population dynamics of C. raciborskii. Best performing models based on using all measured variables as inputs indicated electrical conductivity (EC) within the range of 206-280mSm-1 as threshold above which fast growth and high abundances of C. raciborskii have been observed for the three lakes. Best models based on electronically measurable variables for the Lakes Wivenhoe and Somerset indicated a water temperature (WT) range of 25.5-32.7°C within which fast growth and high abundances of C. raciborskii can be expected. By contrast the model for Lake Samsonvale highlighted a turbidity (TURB) level of 4.8 NTU as indicator for mass developments of C. raciborskii. Experiments with online measured water quality data of the Lake Wivenhoe from 2007 to 2010 resulted in predictive models with 0.61≤r2>0.65 whereby again similar levels of EC and WT have been discovered as thresholds for outgrowth of C. raciborskii. The highest validity of r2=0.75 for an in situ data-based model has been achieved after considering time lags for EC by 7 days and dissolved oxygen by 1 day. These time lags have been discovered by a systematic screening of all possible combinations of time lags between 0 and 10 days for all electronically measurable variables. The so-developed model performs seven-day-ahead forecasts and is currently implemented and tested for early warning of C. raciborskii blooms in the Wivenhoe reservoir.

Increased incidence of Cylindrospermopsis raciborskii in temperate zones--is climate change responsible?

The bloom-forming, toxic cyanobacterium, Cylindrospermopsis raciborskii exhibits global distribution. In recent years both the occurrence and dominance of this species, particularly in temperate regions, has increased. Whilst this may be due to increased sensitivity of analytical detection methods or more rigorous sampling routines, it is possible that this expansion has been assisted by a number of changing conditions in these environments. The geographical expansion of both the organism and toxin production can be attributed to phenomena such as eutrophication and climate change. In this review, we discuss the occurrence of C. raciborskii with respect to current literature against the backdrop of increasing global temperatures. Critically, we identify a concerning trend between the geographical spread of this organism and global climate change.

The impacts of a high CO2 environment on a bicarbonate user: the cyanobacterium Cylindrospermopsis raciborskii.

The potentially toxic cyanobacterium Cylindrospermopsis raciborskii (Woloszynska) Seenya et Subba Raju, originally described as a tropical-subtropical species, is increasingly found in temperate regions and its range is expanding. Climate change is hypothesised to be a factor in this expansion. We studied the effects of dissolved inorganic carbon (DIC) and pH on growth and photosynthesis of this species. We prepared six treatments in a continuous culture (turbidostat) grown at high light, two at low light, and eight in batch cultures grown under low light, by manipulating pH, HCO(3)(-) and CO(2) to assess the effect, if any, of these parameters on the growth rate, inorganic carbon acquisition and photosynthetic parameters of C. raciborskii. When the turbidostats were grown at 100 µmol photons (PAR) m(-2) s(-1), HCO(3)(-) concentration and pH had a positive effect on growth rate; the specific growth rate in 6 mM HCO(3)(-), for example, was twice what it was in 0.6 mM HCO(3)(-) (0.84 ± 0.10 and 0.44 ± 0.04 d(-1) respectively). Growth was lower in turbidostats grown at 20 µmol photons (PAR) m(-2) s(-1). Isotope disequilibrium experiments showed that the contribution of HCO(3)(-) to DIC acquisition is proportionately greater at the higher light. The maximum relative electron transport rate (rETR(max)) was significantly higher at the higher light, while the slope of the linear portion of the rETR(max) versus irradiance curve (α) was unchanged. In low light batch cultures, increasing HCO(3)(-) concentration and pH had a negative effect on growth, while CO(2) concentration had a small, positive effect. This species of cyanobacterium has an efficient CCM and under ideal growing conditions gets most of its carbon from HCO(3)(-). It may, therefore, be at a competitive disadvantage in a high CO(2) world.

Removal of saxitoxins from drinking water by granular activated carbon, ozone and hydrogen peroxide--implications for compliance with the Australian drinking water guidelines.

In a laboratory-scale trial, we studied the removal of saxitoxins from water by ozone, granular activated carbon (GAC) and H(2)O(2), and considered the implications of residual toxicity for compliance with the Australian drinking water standards. Cell-free extracts of Anabaena circinalis were added to raw, untreated drinking water obtained from a water supply reservoir to provide a toxicity of 30 microg (STX equivalents)l(-1). Ozone alone, or in combination with H(2)O(2), failed to destroy the highly toxic STX and GTX-2/3, and only partially destroyed dc-STX, and the low-toxicity C-toxins and GTX-5. In all cases, the toxicity of the water was reduced by less than 10%. GAC removed all of the STX, dc-STX and GTXs, but only partially removed the C-toxins. However, the residual toxicity was reduced to the suggested Australian drinking water guideline concentration of 3 microg (STX equivalents)l(-1) without O(3) pre-treatment. Modelling the spontaneous chemical degradation of residual C-toxins following treatment shows that residual toxicity could increase to 10 microgl(-1) after 11 d due to formation of dc-GTXs and would then gradually decay. In all, residual toxicity would exceed the Australian drinking water guideline concentration for a total of 50 d.

Exposure of beef cattle to sub-clinical doses of Microcystis aeruginosa: toxin bioaccumulation, physiological effects and human health risk assessment.

Yearling beef cattle were fed 1 x 10(5) cells ml(-1) of toxic Microcystis aeruginosa in their drinking water for a period of 28 d. Feed and water intakes of four control and four treated animals remained unchanged following introduction of M. aeruginosa into the drinking water of the treatment animals, and there were no significant differences in feed and water intakes between control and treated animals. We tested the blood plasma of both control and treated animals twice each week for elevated concentrations of the liver enzymes gamma-glutamyl transferase (GGT), glyceraldehyde dehydrogenase (GADH), amino aspartate transferase (AST) and bilirubin. All tests were negative indicating no measurable liver dysfunction resulting from microcystin intoxication. We also tested for the presence of free microcystin in the liver and blood plasma by HPLC and ELISA and for total microcystin (free+bound) in the liver by GC-MS. If all the ingested microcystin was bioaccumulated within the liver, the concentration would have exceeded 3 microg MCYST-LR g(-1) fresh weight. HPLC and GC-MS analysis of the liver tissue and blood plasma from treated animals failed to detect any microcystins. ELISA analysis of liver tissue extracts from the treated animals indicated microcystin concentrations as high as 0.92 microg MCYST-LR equivalents g(-1) fresh weight although none was indicated in the blood plasma. The microcystin concentrations measured by ELISA in livers of treated animals were more than 1000 times higher than the limit of quantification by HPLC and GC-MS indicating the ELISA results were almost certainly due to cross reactivity with something other than intact MCYST-LR. Based on the detection limits of the HPLC and the per capita daily consumption of beef in Australia, it appears that consumption by beef cattle of water containing M. aeruginosa cell concentrations up to 1 x 10(5)cells ml(-1) for 4 weeks would not produce concentrations of microcystin within the liver or blood plasma that would present an unacceptable risk to human health based on World Health Organization protocols for determining such risks.

Eleocharis sphacelata: internal gas transport pathways and modelling of aeration by pressurized flow and diffusion.

The ability of diffusive gas transport and pressurized, convective flow to satisfy internal oxygen demands was examined for an aquatic sedge, Eleocharis sphacelata R. Br. Resistances to convection and diffusion through the plant were quantified from anatomical studies of the airspace dimensions, and these were used in mathematical models to calculate the fluxes required to satisfy oxygen demands measured in the tissue. The greatest resistance to diffusion in the underwater tissue was the submerged culm between the waterline and sediment surface (1560 Ms m-3 per m culm length). Resistances of the nodal intercalary meristem (52 Ms m-3 ) and rhizome internode (34 Ms m-3 ) were minor. In contrast, resistances to convection were low in the culms (38 MPa s m-3 per m culm length), and higher in the nodal meristems (93 MPa s m-3 ). The rhizome internodes had large cortical canals with a low convective resistance (0.75 MPa s m-3 ), and a parallel spongy pith with a very high resistance (518 MPa s m-3 ) that is probably short-circuited by convection. The resistance of the submerged culm means that diffusion is inadequate to satisfy oxygen demands in plants growing in >10 cm of water, and that convection is therefore essential in the natural habitat of this species (water to c. 2 m depth). However, a convective oxygen influx as low as 28 × 10-8 mol s-1 per culm (equivalent to a gas flow rate of 3 µl s-1 per culm) could satisfy the entire oxygen demand of the underwater tissue; this value is well below actual rates. At this flow rate, the spongy pith in the rhizome would also remain aerobic: it has a low resistance to diffusion (73 Ms m-3 ) and could receive sufficient oxygen by diffusion from the node. The data agree well with previous empirical measurements of convection in this species and show that diffusion and convection are both important processes for its aeration.

H+ exchange and nutrient uptake by roots of the emergent hydrophytes, Cyperus involucratus Rottb., Eleocharis sphacelata R. Br. and Juncus ingens N. A. Wakef.

We immersed the root systems of three emergent aquatic monocots (Cyperus involucratus Rottb., Eleocharis sphacelata R. Br. (both Cyperaceae), and Juncus ingens N. A. Wakef. (Juncaceae)) in nutrient agar gels with pH indicators and pH microelectrodes to study H+ exchange by root systems. For all three species, the pH changed at the tips of the adventitious roots and around young laterals. Older, lignified surfaces had little effect on pH. The pH changes around the laterals were affected by the form of nitrogen in the nutrient gel When NO3 - was supplied as the N source, there was a small pH increase, whereas NH4 + enhanced H+ release, causing the pH adjacent to the laterals to decrease below 4.5. In contrast, root tips acidified the media, irrespective of the nutrient composition. H+ exchange was quantified from titrimetric assays of H+ exchange in nutrient solutions. There was a net H+ efflux from the roots of all three species into complete nutrient solutions with NH4 + as the N source, ranging from 246 µmol H+ h-1 g-1 d. wt for C. involucratus, to 32 µmol H+ h-1 g-1 d. wt for J. ingens. The H+ efflux into similar nutrient solutions with NO3 - as the N source ranged from -89 µmol H+ h-1 g-1 d. wt for C. involucratus to -0.8µmol H+ h-1 d. wt for J. ingens. By assuming that H+ exchange was mainly located in the laterals, rates of H+ excretion as high as 542 µmol H+ h-1 g-1 d. wt can be calculated for C. involucratus in NH4 + solutions. Rates of H+ excretion by C. involucratus were scarcely affected by solutions of most Other ions. The NH4 + -induced H+ release was dramatically reduced by H+ pump inhibitors, attaining only 18% of control rates with 50 µM diethylstilbestrol and 38% of control rates with 5 µM dicyclohexylcarbodiimide.