Grantee Research Project Results
Final Report: Ecophysiology Studies of Pseudo-Nitzschia Species
EPA Grant Number: R826306Title: Ecophysiology Studies of Pseudo-Nitzschia Species
Investigators: Wells, Mark , Goldman, Joel C. , Garrison, David L. , Tjeerdema, Ronald S.
Institution: University of California - Santa Cruz
EPA Project Officer: Packard, Benjamin H
Project Period: January 1, 1998 through December 31, 2001
Project Amount: $529,999
RFA: Harmful Algal Blooms (1997) RFA Text | Recipients Lists
Research Category: Water Quality , Water , Aquatic Ecosystems
Objective:
The objective of this research project was to investigate the ecophysiology of toxigenic Pseudo-nitzschia spp. in relation to macronutrient and micronutrient stress imposed in laboratory cultures. The purpose was to identify the conditions most conducive for the production and accumulation of the potent neurotoxin domoic acid (DA) by these diatoms. The project focused on studying the effects of macronutrient stress and micronutrient (trace metal) stress during exponential phase growth of the organisms.Summary/Accomplishments (Outputs/Outcomes):
Determination of DA Concentrations. We implemented and tested two methods for determining particulate DA concentrations; the high performance liquid chromatography/ultraviolet (HPLC/UV) method and the 9-fluorenylmethyl chloroformate (FMOC) method. These tests showed that the HPLC/UV method lacked sufficient sensitivity for the analysis of high cell densities in our cultures, necessitating the use of the more involved FMOC analytical method. While the latter method worked well for particulate samples, it too lacked sufficient sensitivity to quantify the release of DA from cells growing in log phase in the metal manipulation experiments (see below). Therefore, we collaborated with Dr. Eden Rue (UCSC) to measure dissolved (< 0.2 µm) DA using competitive ligand exchange cathodic stripping voltammetry (CLE-CSV). This highly sensitive method measured extremely low (nM) concentrations of DA-equivalents in our culture experiments with synthetic seawater media. DA equivalents are defined here as molecules that have analytically identical conditional stability constants for Fe and Cu as DA. These measurements would not be appropriate in amended natural seawater media due to interference with other, more abundant ligand classes, but we expected this quantification to be accurate in the synthetic media used in these experiments. A subset of the CLE-CSV determinations were validated against a new, highly sensitive DA-specific cELISA method in collaboration with Dr. Neale Towers and Dr. Ian Garthwaite (AgResearch, New Zealand).
Culture Experiments-Macronutrient (Si, P, N) Stress Factors. We reared Pseudo-nitzschia spp. in continuous cultures using both amended natural seawater and synthetic seawater. We tested the effects of macronutrient and Fe limitation on the intracellular accumulation of DA. We analyzed both culture and field samples for DA concentrations using the FMOC method.
Continuous culture experiments were conducted using custom designed and fabricated polycarbonate culture vessels. We tested the effects of Si, P, and N limitation on the accumulation of DA by Pseudo-nitzschia multiseries and Pseudo-nitzschia australis clones isolated from Monterey Bay, CA. These experiments entailed multiple runs at various culture dilution rates. We found poor agreement between measurements of in vivo fluorescence and cell counts, necessitating the time-consuming cell counting approach to quantifying cell growth rates. Samples were retained for cell counts, bacteria counts, nutrients, particulate organic carbon (POC), particulate organic nitrogen (PON), and DA analyses (see above) once cultures had reached-steady state conditions (defined as 3 consecutive days whereby the percent relative standard deviation of cell counts among days varies by less than 10 percent.).
Based on independent dilution series runs for Pseudo-nitzschia multiseries, intracellular DA concentrations increase with increasing Si limitation and P limitation (i.e., lower growth rates), consistent with results reported for east coast P. multiseries isolates. Preliminary evaluations of recent results suggest a similar effect of Si and P limitation on P. australis . The latter experiments are the first evaluation of macronutrient stress on DA production by this organism, which was responsible for the major toxic bloom event on much of the California coastline during May 1998. DA accumulation was not seen under N limitation in any of the toxic species, as has been reported for other Pseudo-nitzschia spp. The inverse relationship between macronutrient- regulated growth rates and DA accumulation suggest that DA production/accumulation may be due more to slower dilution of the cytoplasm (i.e., lower cell division rates) than to any direct mechanistic linkage between Si or P stress on DA production rates.
The timeline of these experiments was behind the targets proposed, due to difficulties in isolating and maintaining P. australis in culture. Both in our laboratory and elsewhere, P. australis has proved difficult to maintain in culture over time. Thus, it often was necessary to re-isolate healthy cultures from field populations. Because of the seasonal appearance of species, it sometimes required several months to complete an experimental run. However, we began progressing well since last summer, when we were finally able to confirm the isolation of healthy mixed clonal isolates of P. australis .
Development of a Synthetic Seawater Medium for Trace Metal Experiments. Trace metal availability to phytoplankton cannot be rigorously and reproducibly regulated in amended seawater media due to variable amounts of metals in different batches of seawater, general metal contamination introduced upon traditional sampling methods, and the poorly understood effects of strong metal-specific chelators in seawater, the concentrations of which can vary among batches with storage time. Developing an appropriate synthetic medium also should help overcome some of the inherent inconsistencies in Pseudo-nitzschia spp. growth in seawater media. Beginning with the basal salt matrix described in Price, et al. (1989), considerable time and effort was expended to test the effect of different macro- and micro-nutrient mixes on growth of Pseudo-nitzschia australis and Pseudo-nitzschia multiseries. Unlike many algal species, which are very tolerant to a range of artificial media, P. multiseries and P. australis are much more sensitive, requiring that trace metal contaminants in the reagent salts be removed by ion exchange. By amending this basal salt solution with different mixtures of macronutrients (N, P, Si), metals (Fe, Mn, Co, Cu, Zn, Se, Mo), chelator (EDTA) and vitamins (thiamine, biotin, and B12), we eventually arrived at an optimal nutrient mixture, which is described in Maldonado, et al., 2002. This medium was employed for all of the trace metal experiments that follow.
Culture Experiments-Micronutrient (Fe, Cu) Stress Factors. We used the synthetic growth medium to conduct a wide range of experiments in which the availability of Fe and Cu were varied, while all other growth factors were kept optimal. Metal ion activities (i.e., availability) were regulated by varying the additions of metal and the chelator EDTA (as calculated by the equilibrium reaction model MINEQL+). Unlike many previous culture studies that focused on cell toxicities in late, or stationary phases of culture growth, we instead chose to study DA production in healthy cell populations in log phase growth. Semi-continuous batch culture methods were used, whereby cell abundance was monitored daily, and cells were transferred to new media every 3-4 days to maintain them in log phase growth. Under these conditions, metal ion activities are buffered by EDTA to maintain uniform metal availabilities; cell transfers are needed to keep macronutrients at optimal concentrations. It also is more logistically straightforward to minimize metal contamination using semi-continuous batch cultures as opposed to continuous cultures.
Using these established methods, we determined that the cellular Fe quotas for optimal growth of P. multiseries and P. australis are similar to those of other coastal diatoms. However, normalized to surface area, Fe uptake rates are approximately five times less efficient, meaning that these toxigenic diatoms should become stressed by Fe availability before other coastal phytoplankton. We have used additions of Desferal (a fungal siderophore) in field incubations to support the contention that these toxigenic species are more challenged by low Fe availability in coastal waters than other coastal diatoms.
Both Cu and Fe stress greatly increase (10-20 times) the production rate of DA by P. multiseries and P. australis during log phase growth. However, this additional DA is not retained by the cell; instead, it is released to the surrounding medium, with intracellular residence times of DA being reduced from approximately 14 hours to about 40 minutes moving from Fe-replete to Fe-deficient conditions. Accumulation of DA intracellularly occurs only when cell growth slows, as seen in the Si and P limited cultures. It is possible that DA production is constitutive (i.e., a constant feature) causing cell concentrations to increase as dilution of cytoplasmic components decreases with lower cell division rates.
The ecological advantage imparted to toxigenic Pseudo-nitzschia spp. by releasing DA to seawater is unclear. Our experiments show that adding DA partially alleviates Cu toxicity in cultures. However, the Cu tolerance of Pseudo-nitzschia spp. determined in this study indicate that the free cupric ion activities measured in typical coastal waters lie well within the range for their optimal growth. It seems difficult to ascribe a major role for Cu in affecting the DA toxicity during later stages of Pseudo-nitzschia blooms; Cu toxic conditions would prevent the buildup of the population. Nonetheless, it is conceivable that Cu may regulate cell toxicities in early bloom stages.
On the other hand, it is much easier to visualize how the effect of Fe on toxin production and release could affect bloom toxicity. Perhaps this could explain the occurrence of high numbers of toxigenic Pseudo-nitzschia with little or no presence of particulate toxins. Because DA significantly improves Fe uptake rates over the otherwise poor rates observed for inorganic Fe species, its release may or may not be required at the peak of bloom development, depending on the relative availability of Fe in seawater (low Fe-DA release and low-toxicity cells; high Fe-no DA release and high-toxicity cells).
Due to auspicious circumstances, we were able to conduct bioassay experiments during the massive P. australis bloom in Monterey Bay in 1998 (an NSF-funded cruise). Samples were collected near the peak of the bloom from a number of stations within the bay from two depths: 5 m and the depth of the chlorophyll maximum. In addition to characterizing the water column structure with numerous CTD profiles, samples were retained for nutrients, chlorophyll, trace metals, cell counts, and particulate DA determinations. Using on-deck incubations, we were able to show that these toxigenic diatoms became more easily Fe stressed than non-toxigenic diatoms outside the bloom region. Moreover, by adding the fungal siderophore (Desferal) to decrease Fe availability to Pseudo-nitzschia australis in natural population cultures, cellular DA concentrations fell by over an order of magnitude. These results are consistent with the laboratory findings that decreased Fe availability caused a decrease in cellular DA concentrations.
Synthesis of these laboratory and field data suggests that Fe availability is a key factor in determining whether blooms of toxigenic diatoms become toxic or not. Our findings suggest that the primary biochemical role of DA lies outside the cell, rather than within the intracellular metabolism, and is related to facilitating Fe uptake. If so, toxic bloom conditions will result when cells become limited by factors other than metal stress, thus allowing the buildup of internal toxin reserves. However, if Fe (or perhaps Cu) stress occurs in conjunction with macronutrient (or other) limitation, the cells will transport DA to the surrounding environment, depleting their toxin content, and thus reducing the toxicity of filter feeders or planktivorous fish feeding upon these algae. An example of the latter case may be the massive Pseudo-nitzschia bloom that occurred offshore of Monterey Bay, CA, in August 2000. This bloom, advected offshore into a region known to have low Fe availability, had extremely low cell toxicities. This project has resulted in a significantly improved understanding of the physiological control of DA production by these toxigenic diatoms, and has opened a new series of hypotheses for the metabolic role of DA in the cell and the factors that determine whether or not blooms become toxic when they occur.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 11 publications | 1 publications in selected types | All 1 journal articles |
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Type | Citation | ||
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Maldonado MT, Hughes MP, Rue EL, Wells ML. The effect of Fe and Cu on growth and domoic acid production by Pseudo-nitzschia multiseries and Pseudo-nitzschia australis. Limnology and Oceanography 2002;47(2):515-526. |
R826306 (2000) R826306 (Final) |
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Supplemental Keywords:
human health, organism, chemicals, toxics, metals, environmental chemistry, biology, ecology, measurement methods, northeast, northwest., RFA, Scientific Discipline, Water, Geographic Area, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, Ecosystem/Assessment/Indicators, Ecosystem Protection, State, Oceanography, Ecological Effects - Environmental Exposure & Risk, algal blooms, Pacific Northwest, Biology, seabird deaths, aquatic, aquatic ecosystem, ecological effects, ecological exposure, bloom dynamics, estuaries, Pseudo-Nitzschia species, Oregon, harmful algal blooms, nutrient kinetics, Washington (WA), transport and concentration, iron deficient conditions, ecophysiology, California (CA), domoic acid producing diatoms, ORProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.