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The Influence of Trace Metals and Zebra Mussels on Microcystis Growth and Toxin Production
Primary Investigator:
Donna Kashian - NOAA/GLERL
Co-Investigators:
Juli Dyble Bressie - NOAA/GLERL
Chuanwu Xi, Jerome Nriagu - School of Public Health,
University of Michigan*
Executive Summary of Rationale
Recent increases in the toxic cyanobacterial HAB Microcystis pose a significant threat to water quality and resource utilization in the Great Lakes. Thus there is great need for the capacity to predict the timing, movement and magnitude of Microcystis blooms in order to minimize detrimental ecological and human health impacts. While much is known about cyanobacterial ecology, critical questions remain regarding the environmental factors controlling the production of microcystin for the Great Lakes. Our objective is to investigate the role of trace metals and zebra mussels on the growth of Microcystis and their toxin production. Our approach will be to: 1) develop flow cytometry and fluorescence in situ hybridization (FISH) methods for measuring the concentration of toxic Microcystis using microcystin synthetase genes; 2) use these methods to cross validate the quantitative PCR method already developed for quantifying toxic Microcystis; 3) determine the binding capacity and bioavailable levels of copper and zinc in areas of Saginaw Bay with and without zebra mussels; and 4) determine if there is a relationship between the concentration of toxic Microcystis, expression of microcystin synthetase genes and the concentrations (bioavailable levels?) of trace metals (mainly iron, copper and zinc) in the Bay.
Proposed Work
Current/Ongoing
Currently, our best method of quantifying the number of toxic Microcystis cells is quantitative PCR, which amplifies the number of copies of the mcyB gene from extracted cells and quantifies the amount present in a sample based on a standard curve. This method works well for determining relative concentrations of toxic cells between samples, but can be biased by different efficiencies in extracting the DNA from cells, multiple copies of the gene in a single cell, amplification biases inherent in PCR and rough estimations of the number of cells in a colony. Therefore, we propose to begin to develop a new methodology for quantifying toxic Microcystis in collaboration with Dr. Xi at the UM School of Public Health (SPH). Flow cytometry is a method often used to count bacterial cells, in which a narrow stream of cells is passed through a beam of light and the scattering properties of the light are detected optically and related to cell volume and concentration. This protocol has not traditionally been used with Microcystis because the large size of the colonies would clog the intake orifice of the cytometer. However, recent success in using sonication to break the colony into individual cells will allow us to use this technique for quantification. The other challenge is to not only count the number of Microcystis cells, but to quantify how many are toxic. To do this, we will use fluorescent in situ hybridization (FISH), which uses fluorescent probes that are designed to bind to the mcyB gene. The Microcystis cell wall will be chemically-induced to be more permeable, allowing the probe to enter the whole cell and bind to mcyB if the cell is toxic, producing a fluorescent signal. Then the Microcystis cells can be run through the flow cytometer, which will quantify the number of toxic vs. non-toxic cells based on fluorescence. This could be an incredibly powerful way to accurately quantify toxic cells in both environmental and experimental samples.
In addition to technique development, the other primary goal of this program is to assess how dreissenid mussels may transform key trace metals to make them more bioavailable and thus impact cyanobacterial growth. In the field portion of this project, the correlation between trace metal availability and occurrence and toxicity of Microcystis will be assessed by collecting water samples from 4-5 sampling locations at various depths in Saginaw bay in June and August 2008. Trace metal (iron, copper and zinc) concentrations and bioavailability will be measured and the number of toxic Microcystis cells will be quantified by both flow cytometry with FISH and quantitative PCR. In order to determine the binding capacity and bioavailable levels of copper and zinc in areas of Saginaw Bay with and without zebra mussels, we will also collect water immediate above a zebra mussel bed and a similar distance from the substrate in a location where zebra mussels are not present, such as a deeper region in the bay, and do the same set of analyses.
To further understand the relationship among zebra mussels, concentration of toxic cyanobacteria, and the concentrations of trace metals (mainly iron, copper and zinc), we will coordinate with experiments led by Hank Vanderploeg. He will be conducting lab experiments to assess the impact of zebra mussel feeding on the concentration and toxicity of Microcystis. We will collect samples at the beginning and conclusion of his zebra mussel feeding experiments from the beakers with and without zebra mussels and measure changes in trace metal bioavailability and concentration of toxic Microcystis using quantitative PCR and flow cytometry with FISH. In combination with the data from Vanderploeg on these experiments, we hope to assess whether changes in trace metal bioavailability due to dreissenid grazing may play a role in promoting cyanobacterial blooms in the Great Lakes.
Scientific Rationale
Recent harmful algal blooms (HABs) in Saginaw Bay, Lake Huron, have caused considerable concern because this region is largely dependent on the bay for public water supply and economic revenue through recreational use of beaches, fishing, and boating. The proliferation of these undesirable algae is one of the driving factors in designating Saginaw Bay as an Area of Concern (and is largely the result of nutrient inputs from its extensive watersheds). High algal biomass has resulted in taste and odor complaints in drinking water and concerns about algal toxins produced during bloom events can result in animal and human illness (Hawkins et al 1985, Teixera et al. 1993, Kuiper-Goodman et al. 1999). The common toxin produced and released by cyanobacteria in Saginaw Bay is microcystin. Recent studies have measured up to 5 µg/L intracellular microcystin (Dyble et al, accepted), exceeding the recommended limit for microcystin in drinking water (1 µg/L; World Health Organization 1998). These microcystin concentrations are of particular concern because they are found close to a public water supply intake in the bay. The degradation of algal blooms washed up onto the shoreline can also reduce the aesthetic value of the beaches around Saginaw Bay. Although cyanobacterial bloom events have occurred historically in the Great Lakes, their widespread recurrence within the last 10 years is thought to be related to the introduction of dreissenid mussels to the system and has resulted in a significant threat to ecosystem and human health.
Despite the widespread abundance of Microcystis, limited information on the environmental factors controlling the production of microcystin exists for the Great Lakes. Recent studies have challenged the prevailing paradigm that microcystin is produced as a secondary metabolite, and suggest that microcystin production is tightly coupled to cell growth and division (Orr & Jones 1998). Thus environmental drivers such as nutrients and light may be key factors in the prediction of Microcystis bloom toxicity. Furthermore, cyanobacterial interactions with metals have been frequently reported, but seldom in the bloom-forming context. It was suggested that cyanobacteria produce toxins as a mechanism to cope with environmental stress (Paerl, 1996). Trace metals elicit a variety of acute and chronic toxicity effects (Heng et al., 2004; Miao et al., 2005) and cyanobacteria have some capability to accumulate, detoxify, or metabolize such contaminants (Feris et al., 2004; Garcia-Meza et al., 2005; Wang et al., 2005). For example, microcystins can bind several cations such as iron, copper and zinc, which provide the molecules siderophoric (chelating) properties (Humble et al., 1997). Microcystins as siderophores may bind Fe2+ from the environment for use by the cell under iron-limited conditions (Utkilen and Gjolme 1995). Under conditions of high cellular iron concentrations, an intracellular siderophore may have a protective function by chelating ferrous iron and keeping the free iron low (Lukac and Aegerter 1993). Therefore, iron-limited or iron-depleted environments may favor the growth of microcystin-producing cyanobacteria. The use of algaecides, such as copper sulphate, to control cyanobacterial growth in water supply reservoirs is a common procedure, but Cu-resistant cells have arisen by spontaneous mutations (Garcia-Villada et al. 2004) and, furthermore, microcystins may also contribute to the survival and growth of microcystin-producing cyanobacteria under such conditions. However, there is no systematic study of the impact of trace metals on triggering toxic cyanobacterial blooms and toxin production in the environment. Dreissenid mussels may alter the bioavailability of trace metals including Fe, Cu and Zn and thus this may be a possible mechanism by which their introduction has promoted Microcystis blooms in invaded systems. The field study is designed to determine if there is a correlation between the presence of dreissenids, trace metal concentration and availability, microcystin and abundance of toxic Microcystis.
Governmental/Societal Relevance
Harmful algal blooms are of great importance to NOAA, the scientific community and the public due to their potentially significant detrimental impact on ecosystem and human health. In light of this, mandates for their study have come from both the legislature and the scientific community. The widespread presence of microcystin concentrations above the WHO recommended limit of 1 µg L-1 in western Lake Erie and Saginaw Bay stresses that detection of toxic Microcystis blooms and understanding the mechanisms stimulating toxin production are highly relevant to both the Great Lakes community and to GLERL. Traditional measures of detecting Microcystis by microscopic cell counts have proven insufficient due because the time required for analysis is not conducive to rapid management decisions and cell counts are not an accurate indicator of toxicity. Microcystis blooms may be comprised of both toxic and/or nontoxic strains and a largely non-toxic bloom will not likely have a significant human health risk. The proposed research will develop methods for detecting toxic strains of Microcystis and determining water chemistry factors that may influence blooms formations in Saginaw Bay.
Relevance to Ecosystem Forecasting
This project has direct links to forecasting the effects of toxic Microcystis blooms in the lower Great Lakes. Microcystis blooms threaten human health when they produce toxins. So in order to develop forecasts for the presence of toxic blooms, it is essential to know what environmental and ecological factors contribute to Microcystis growth and toxin production. The results of this research can provide input for a biophysical model than will couple Microcystis growth and toxin production to basin hydrodynamics to provide forecasts that will be useful for monitoring and managing toxic blooms.
Cited References
Dyble, J., Fahnenstiel, G., Litaker, W., Millie, D.and Tester, P., accepted. Microcystin concentrations and genetic diversity of Microcystis in Saginaw Bay and western Lake Erie. Environmental Toxicology.
Feris KP, Ramsey PW, Frazar C, et al. (2004) Seasonal dynamics of shallow-hyporheic-zone microbial community structure along a heavy-metal contamination gradient. Appl Environ Microbiol 70, 2323-2331.
Garcia-Meza JV, Barrangue C, Admiraal W (2005) Biofilm formation by algae as a mechanism for surviving on mine tailings. Environ Toxicol Chem 24, 573-581.
Garcia-Villada L, Rico M, Altamirano MM, et al. (2004) Occurrence of copper resistant mutants in the toxic cyanobacteria Microcystis aeruginosa: characterization and future implications in the use of copper sulphate as algaecide. Water Res 38, 2207-2213.
Heng LY, Jusoh K, Ling CH, Idris M (2004) Toxicity of single and combinations of lead and cadmium to the cyanobacteria Anabaena flos-aquae. Bull Environ Contam Toxicol 72, 373-379.
Humble AV, Gadd GM, Codd GA (1997) Binding of copper and zinc to three cyanobacterial microcystins quantified by differential pulse polarography Water Res 31, 1679-1686.
Lukac M, Aegerter R (1993) Influence of trace metals on growth and toxin production of Microcystis aeruginosa. Toxicon 31, 293-305.
Kuiper-Goodman, T., I. Falconer, and J. Fitzgerald. 1999. Human health aspects. In: Toxic Cyanobacteria in Water: A guide to their public health consequences, monitoring and management (Chorus I, Bartram J, eds). London:Spon, 113-153.
Miao H, Jiang L, Huang L (2005) Effects of simvastatin on the expression of intercellular adhesion molecule-1 mRNA in neonatal brain with hypoxic-ischemic damage. J Nanosci Nanotechnol 5, 1261-1265.
Orr, P.T.and Jones, G.J., 1998. Relationship between microcystin production and cell division rates in nitrogen-limited Microcystis aeruginosa cultures. Limnol. Oceanogr. 43: 1604-1614.
Teixera, M.G.L.C., Costa, M.C.N., Carvalho, V.L.P., Pereira, M.S. and Hage, E., 1993. Gastroenteritis epidemic in the area of the Itaparica Dam, Bahia, Brazil. Bulletin of the Pan American Health Organization 27, 244-253.
Utkilen, H. and N. Gjolme. 1995. Iron-stimulated toxin production in Microcystis aeruginosa, Appl. Environ. Microbiol. 61:797-800.
WHO, 1998. Cyanobacterial toxins: microcystin-LR. In: Guidelines for drinking water quality. 2nd Edition, Addendum to Vol 2. Health criteria and other supporting information. World Health Organization, Geneva, Switzerland, pp. 95-110.
*Link leads off GLERL's website
