Grantee Research Project Results
2000 Progress 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 Period Covered by this Report: January 1, 1999 through December 31, 2000
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 is to investigate the ecophysiology of toxigenic Pseudo-nitzschia spp. in relation to macronutrient and micronutrient stress effects on the production and accumulation of domoic acid (DA).Progress Summary:
We are rearing Pseudo-nitzschia spp. in continuous cultures and in semi-continuous batch cultures using both amended natural seawater and synthetic seawater. We are currently testing the effects of Si limitation and Fe limitation on the intracellular accumulation of DA. We have analyzed both culture and field samples for domoic acid concentrations using the FMOC method.
Continuous culture experiments are being conducted using custom designed and fabricated polycarbonate culture vessels. We have tested the effects of Si and P limitation on the accumulation of DA by Pseudo-nitzschia multiseries and Pseudo-nitzschia australis clones isolated from Monterey Bay, California. These experiments have entailed multiple runs at numerous 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 are retained for cell counts, bacteria counts, nutrients, POC, PON, and domoic acid analyses (see below) once cultures have reached steady state conditions (defined as 3 consecutive days whereby the percent RSD 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. We now are midway through testing the effect of Si 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. The timeline of these experiments are somewhat behind the targets proposed due to difficulties in isolating P. australis in culture. However, we are progressing well since last fall when we were finally able to confirm the isolation of healthy mixed clonal isolates of P. australis.
We have successfully implemented and tested two methods for determining particulate domoic acid concentrations, the HPLC/UV method and the FMOC method. Initial efforts showed the HPLC/UV method lacked sufficient sensitivity even for the high cell densities in our cultures, necessitating the use of the more involved FMOC determination method. Nonetheless, current limitations of the FMOC method hinder the study of domoic acid release from the cells to the media (see below). Therefore, we have collaborated with Dr. Eden Rue to measure DA using competitive ligand exchange cathodic stripping voltammetry. This highly sensitive method can measure extremely low (nM) concentrations of DA in our culture experiments with synthetic seawater media (see below), but lacks the specificity needed for DA determinations in amended seawater media. We also have collaborated with Dr. Ian Garthwaite (AgResearch, New Zealand) to use a newly developed ELISA method for DA determinations in our culture media.
To overcome the inherent inconsistency in rearing toxigenic Pseudo-nitzschia spp. in enriched seawater mediaresults are highly dependent on the specific source of seawater for unknown reasonswe have developed an artificial seawater media for conducting growth experiments under well-defined metal conditions. Unlike many algal species, which are very tolerant to a range of artificial media, P. multiseries and P. australis have proved to be much more sensitive, requiring that trace metal contaminants in the reagent salts be removed by ion-exchange. By amending this basal salt solution with a range of macronutrients, metals (Fe, Mn, Co, Cu, Zn, Se, Mo), chelator (EDTA) and vitamins (Thiamine, Biotin and B12), we have arrived at a basal medium where the growth rates of P. multiseries are close to maximum rates obtained by f/2 enrichments of Monterey Bay seawater (ca. 1.2 d-1).
We have used this synthetic medium to conduct a wide range of experiments in which the availability of Fe and Cu are varied while all other growth factors are kept optimal. Metal ion activities (i.e., availability) are regulated by varying the additions of metal and EDTA (chelator) (as calculated by the equilibrium reaction model MINEQL+). These experiments employ semi-continuous batch cultures, whereby cell abundance is monitored daily and cells are transferred to new media every 3-4 days to maintain them in log phase growth. This culturing approach provides near uniform nutrient conditions while making it logistically straightforward to minimize metal contamination. These studies are in contrast to many earlier studies that concentrated on the intracellular DA concentrations during cell senescence.
Using these established methods, we have determined that the cellular Fe requirement for growth of P. multiseries and P. australis are similar to that of other coastal diatoms. However, normalized to surface area, Fe uptake rates are ~ 5 x less efficient, meaning that these toxigenic diatoms should become stressed by Fe availability before other coastal phytoplankton. We have use 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 (10-20x) the production rate of DA by P. multiseries and P australis during log phase growth. However, these experiments show that this increased production is directed to outside the cell, with intracellular residence times of DA being reduced from ~14 hours to ~40 minutes. in Fe replete and Fe deficient conditions, respectively. Accumulation of DA intracellularly occurs only when cell growth slows, as seen in the Si and P limited cultures. It is possible then that DA production is constitutive (that is, 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. Additions of DA to toxic Cu conditions partially alleviates Cu toxicity, but the free cupric ion activities measured in typical coastal waters lie well within the range for optimal growth of these species. It seems unlikely then that DA release to control Cu toxicity is needed. More importantly though, additions of DA to solution significantly improve Fe uptake rates over the otherwise poor rates observed for inorganic Fe species. These findings imply that the primary function of DA is to modulate metal (probably Fe) availability to the cell.
Due to auspicious circumstance, we were able to conduct bioassay experiments during the massive P. australis bloom in Monterey Bay (1998) (a NSF-funded cruise Wells). 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 domoic acid 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, and that additions of a fungal siderophore (Desferal) cause a dramatic decrease in cell toxicity over a 3 day on-deck incubation study. These results are consistent with the laboratory findings that decreased Fe availability causes a decrease in cellular DA concentrations.
Synthesis of these laboratory and field data suggest that Fe availability is a key factor in determining whether blooms of toxigenic diatoms become toxic or not (e.g, a massive Pseudo-nitzschia bloom offshore of Monterey Bay, California in August 2000 had very low toxicity). These findings indicate 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, then 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, then the cells will transport DA to the surrounding environment, deplenishing their toxin content and thus reducing the toxicity of filter feeders or planktivorous fish feeding upon these algae.
The work to date is keeping us more or less on the time line proposed in the original project schedule.
Future Activities:
Work on the continuous cultures will proceed using the equipment and vessels in hand. We will continue investigating the effects of Si and P stress on the growth and production of domoic acid by P. multiseries and P. australis. We also intend to continue testing the effect of metal stress on the toxin content of these species. The effect of short term Fe depletion on the DA content of actively growing cells in chemostats will be measured. Desferal additions will be used to diminish Fe availability in these pulse/chase type experiments. In addition, we will similarly increase Cu additions to generate chronically toxic levels in chemostats stabilized under mild Si and P stress. The intent of these experiments will be to ascertain whether metal stress indeed has a more dramatic effect on cell toxin contents than does macronutrient nutrition.
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 |
---|
Type | Citation | ||
---|---|---|---|
|
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) |
Exit Exit |
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.