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Genetic and environmental factors influencing Microcystis bloom toxicity
Collaborators
Dr. Gary Fahnenstiel
Dr. Wayne Litaker, NOAA, Beaufort NC
Executive summary
Microcystis community structure and microcystin production are controlled by both environmental factors (such as light, nutrients, grazing pressure) and genetic composition. Essential for forecasting blooms of toxic Microcystis is the ability to determine the toxicity of a bloom, which requires knowing both the number of toxic genotypes present and environmental regulators of toxin production. Cell counts are commonly used to quantify the magnitude of a Microcystis bloom, but are not generally an accurate measure of bloom toxicity due to the presence of both toxic and non-toxic strains. A multiplex PCR assay has been developed to determine the proportion of toxic genotypes from isolated colonies and will be applied to samples collected during the August 2005 and 2006 OHH cruises to see if this method has a good correlation with microcystin concentration. A quantitative PCR method based on a gene involved in microcystin synthesis (mcyB) will be developed to quantify the number of toxic cells present without the biases of colony isolation present in the multiplex PCR. This quantitative PCR method will be applied to samples collected biweekly in western Lake Erie during the summer 2006 to determine the temporal variability in the proportion of toxic genotypes over the progression of a bloom. The expected results of this project will be useful in developing models for forecasting the growth and toxin production in Microcystis blooms in the Great Lakes.
Proposed work
Identify the proportion of toxic genotypes in environmental Microcystis
blooms
During the OHH synoptic cruise in August 2005, samples were collected
for analysis of intracellular and extracellular microcystin concentrations
at 40 stations in western Lake Erie and Saginaw Bay. We will measure the
microcystin concentrations in both the cellular and dissolved fractions
using an antibody-based ELISA kit (according to the protocol in Dyble
et al., submitted). At 14 of these stations, 12-20 Microcystis colonies
were isolated and frozen for genetic analysis. The DNA in each of these
colonies will be extracted and analyzed by multiplex PCR (Dyble et al.,
submitted). This assay will determine if each colony is Microcystis (as
opposed to a morphologically-similar colonial cyanobacteria) and if it
contains the mcyB gene and is thus capable of microcystin production.
The percentage of toxic colonies at each station will be compared to the
microcystin concentrations to evaluate whether this assay is a good indication
of the toxicity of a bloom, as indicated by preliminary data.
Develop a quantitative PCR method for enumerating toxic Microcystis
colonies
We will develop a quantitative PCR (Q-PCR) method for determining the
proportion of toxic genotypes in a Microcystis bloom community. This method
will be based on a similar protocol currently under development for the
toxin genes in another cyanobacterial HAB species Cylindrospermopsis
raciborskii. Genetic sequences of the mcyB gene for Microcystis
strains originating from western Lake Erie and Saginaw Bay are available
through previous work on the OHH project and will be used to design probes
specific to toxic strains of Microcystis. Toxic and nontoxic cultures
of Microcystis aeruginosa that were isolated from western Lake
Erie and Saginaw Bay in August 2005 will be used to validate the assay.
Wayne Litaker (NOAA, Center for Coastal Fisheries and Habitat Research,
Beaufort, NC) is a molecular biologist with extensive experience in developing
molecular techniques for the detection of HAB species and will provide
advice in the development of this assay.
Determine temporal variability in the proportion of toxic genotypes
in western Lake Erie
In addition to determining the spatial variation in the proportion of
toxic Microcystis colonies from the August 2005 cruise, we will
also sample three locations on a biweekly basis during summer 2006 to
determine the temporal variability in the proportion of toxic genotypes.
There is some evidence that there are more toxic genotypes present earlier
in the summer, while nontoxic Microcystis genotypes become more
abundant later in the bloom progression (Janse et al. 2004). However,
in some lakes, microcystin concentrations per cell were the highest in
the late summer, which would not be expected if nontoxic genotypes were
dominant. The relationship between the proportion of toxic genotypes and
microcystin concentrations is still poorly understood, and yet essential
for predicting when Microcystis blooms will present a hazard to
human health in the Great Lakes. The biweekly sampling that is required
to better understand this seasonal variability will be conducted at the
Toledo Light station in western Lake Erie and two additional stations
further out in western Lake Erie in order to sample across a trophic gradient.
This region of western Lake Erie is considered a potential initiation
site for cyanobacterial blooms due to the nutrient inputs from the Maumee
River and thus will be a good location for monitoring the progression
from pre- to post-bloom. This sampling will be done in conjunction with
the sampling for microcystin concentrations on the OHH-Microcystins in
the Great Lakes proposal (Fahnenstiel-04) and will contribute data to
the project on the role of zebra mussels in promoting Microcystis
blooms and toxin production (Dyble-02).
Scientific rationale
The recent increases in cyanobacterial HABs in the Great Lakes has caused significant concern for human and ecosystem health due to the production of toxins by bloom species. In the Great Lakes, Microcystis dominates the cyanobacterial bloom community and produces the hepatotoxin microcystin (Brittain et al. 2000, Carmichael 1994, 1997, Vanderploeg et al. 2001). Preliminary studies have documented the presence of microcystins in the Great Lakes, at times exceeding the recommended limit of 1 µg L-1 of microcystin established by the World Health Organization for drinking water supplies (Brittain et al. 2000, Vanderploeg et al. 2001). The increase in large Microcystis blooms in recent years has caused considerable concern due to the dependence on these waters as a resource and the health risks attributable to microcystins. The ability to accurately measure the distribution and concentration of microcystin in the Great Lakes, including the various microcystin congeners, is therefore essential to protecting human and ecosystem health in this region.
Despite very limited studies, relatively high concentrations of microcystin have been found in Saginaw Bay, western Lake Erie, and in the western region of Lake Ontario (Brittian et al. 2000; Vanderploeg et al. 2001; Murphy et al. 2003). Microcystin concentrations of 3.5 µg L-1 were measured in Saginaw Bay in July 1995, and estimated to be as high as 24 µg L-1 in western Lake Erie in September 1995 (Vanderploeg et al. 2001). In a shallow harbor in western Lake Ontario, microcystin concentrations were as high as 400 µg L-1 in the summer of 2001 (Murphy et al. 2003). In 2003, microcystin concentrations throughout Saginaw Bay and western Lake Erie were commonly above 1 µg L-1 and as high as 58 µg L-1 in wind-accumulated scums (Dyble et al. submitted). Thus, concentrations of microcystin in the Great Lakes may pose a threat to human and ecosystem health.
The production of microcystins by Microcystis and other related cyanobacterial species is under complex genetic and ecological control. Microcystin production is controlled by the mcy genes, a bidirectionally transcribed complex of 10 open reading frames that control synthesis of polyketide synthases and peptide synthetases involved in microcystin biosynthesis (Dittman et al. 1997, Kaebernick et al. 2002). Not all Microcystis strains produce microcystin and the non-toxic strains do not appear to carry the mcy genes. For toxic strains, genetic differences within mcy can result in the production of various amounts and congeners of microcystin. Similar microcystin biosynthesis genes are found in Microcystis, Anabaena and Planktothrix (Christiansen et al. 2003, Rouhiainen et al. 2004). Studies of microcystin cell quota in strains which carry the mcy genes indicate that microcystins are constitutively produced (Kaebernick et al. 2000) and that cell quotas vary with cell growth (Orr and Jones 1998) and between strains (Carmichael 1997). Changes in environmental factors that regulate growth, such as nutrients and light, can result in a 2-10 fold increase in toxicity. These environmentally induced changes in toxicity, however, can be relatively minor in comparisons to the 10-1000 fold increase in bloom toxicity associated with shifts in community composition toward the predominance of more toxic strains (Zurawell et al. 2005). These results imply that the underlying genetic structure of the population will profoundly affect the toxicity of individual blooms and that predicting bloom toxicity requires an understanding of the genetic variation within the bloom and cannot be predicted based on cell counts alone.
Currently, the genetic diversity and the frequency of toxic genotypes are not known for Microcystis blooms in the Great Lakes. In other systems dominated by Microcystis, strains with and without the genetic potential for microcystin production are typically present (Kurmayer et al. 2002, Welker et al. 2003). Preliminary evidence from August 2004 shows that populations of Microcystis in Saginaw Bay and western Lake Erie are composed of both toxic and nontoxic strains. Multiple Microcystis colonies were isolated from 3 stations (2 in Saginaw Bay and 1 in western Lake Erie) and multiplex PCR was used to identify the number colonies positive for mcyB. Of the 40 colonies from Saginaw Bay that were identified as Microcystis using ITS-specific primers, 90% also contained the mcyB gene. Of the 16 Microcystis colonies from western Lake Erie, only 25% contained mcyB. Stations in which there was a higher percentage of toxic colonies also had higher microcystin concentrations. To further investigate this correlation, multiple colonies (15-20) were isolated from 14 stations in western Lake Erie and Saginaw Bay during the August 2005 sampling cruise. The percentage of toxic genotypes in each station will be determined by multiplex PCR and compared to microcystin concentrations to determine if this correlation holds true.
Quantitative PCR is quickly becoming one of the best ways for comparing environmental effects on the genetic level. Combining a measure of the number of mcyB genes (which can be correlated to the number of toxic Microcystis cells present) and the expression of these genes provides a unique way of understanding the impacts of changing environmental parameters on a cellular level. This method can provide information on how the number of toxic genotypes changes over the course of a bloom and be used for determining the distribution of toxic vs. non-toxic strains, neither of which can be determined morphologically and which are essential to our understanding of Microcystis bloom dynamics. The development of this method requires careful design of both the PCR probes and the positive and negative controls. We have developed this method for another cyanobacterial HAB species and will adapt it for Microcystis using the newly purchased Applied Biosystems 7900 Thermal Cycler. We will apply this method using samples collected biweekly in western Lake Erie to answer an important question about the proportion of toxic genotypes during the progression of a Microcystis bloom and also to understanding the role of zebra mussels in the induction of microcystin production. Once this assay has been developed and tested, it could be important in the forecasting of toxic blooms by providing information on when a Microcystis bloom is composed of genotypes that will present a threat to human and ecosystem health.
References Cited
Brittain, S.M., Wang, J., Babcock-Jackson, L., Carmichael, W.W., Rinehart, K.L.and Culver, D.A., 2000. Isolation and characterization of microcystins, cyclic heptapeptide hepatotoxins from a Lake Erie strain of Microcystis aeruginosa. J. Great Lakes Res. 26: 241-249.
Carmichael, W.W., 1994. The toxins of cyanobacteria. Sci. Am. 270: 78-86.
Carmichael, W.W., 1997. The cyanotoxins. Advances in Botanical Research 27: 211-240.
Christiansen, G., Fastner, J., Erhard, M., Borner, T.and Dittmann, E., 2003. Microcystin biosynthesis in Planktothrix: genes, evolution, and manipulation. J. Bacteriol. 185: 564-572.
Dittmann, E., Neilan, B.A., Erhard, M., von Dohren, H.and Borner, T., 1997. Insertional mutagenesis of a peptide synthetase gene that is responsible for heptatoxin production in the cyanobacterium Microcystis aeruginosa PCC 7806. Molecular Microbiology 26: 779-787.
Dyble, J., Fahnenstiel, G., Litaker, W., Millie, D.and Tester, P., submitted. Microcystin concentrations and genetic diversity of Microcystis in Saginaw Bay and western Lake Erie. Environmental Health Perspectives.
Janse, I., Kardinaal, W.E.A., Meima, M., Fastner, J., Visser, P.M.and Zwart, G., 2004. Toxic and nontoxic Microcystis colonies in natural populations can be differentiated on the basis of rRNA gene internal transcribed spacer diversity. Appl. Environ. Microbiol. 70: 3979-3987.
Kaebernick, M., Neilan, B.A., Borner, T.and Dittmann, E., 2000. Light and the transcriptional response of the microcystin biosynthesis gene cluster. Appl. Environ. Microbiol. 66: 3387-3392.
Kaebernick, M., Dittmann, E., Borner, T.and Neilan, B.A., 2002. Multiple alternate transcripts direct the biosynthesis of microcystin, a cyanobacterial toxin. Appl. Environ. Microbiol. 68: 449-455.
Kurmayer, R., Dittmann, E., Fastner, J.and Chorus, I., 2002. Diversity of microcystin genes within a population of the toxic cyanobacterium Microcystis spp. in Lake Wannsee (Berlin, Germany). Microb. Ecol. 43: 107-118.
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.
Rouhiainen, L., Vakkilainen, T., Siemer, B.L., Buikema, W., Haselkorn, R.and Sivonen, K., 2004. Genes coding for hepatotoxic heptapeptides (microcystins) in the cyanobacterium Anabaena strain 90. Appl. Environ. Microbiol. 70: 686-692.
Vanderploeg, H.A., Liebig, J.R., Carmichael, W.W., Agy, M.A., Johengen, T.H., Fahnenstiel, G.L.and Nalepa, T.F., 2001. Zebra mussel (Dreissena polymorpha) selective filtration promoted toxic Microcystis blooms in Saginaw Bay (Lake Huron) and Lake Erie. Canadian Journal of Fisheries and Aquatic Sciences 58: 1208-1221.
Welker, M., Von Dohren, H., Tauscher, H., Steinberg, C.E.W.and Erhard, M., 2003. Toxic Microcystis in shallow lake Muggelsee (Germany) - dynamics, distribution, diversity. Arch. Hydrobiol. 157: 227-248.
Zurawell, R.W., Chen, H., Burke, J.M.and Prepas, E.E., 2005. Hepatotoxic cyanobacteria: A review of the biological importance of microcystins in freshwater environments. Journal of Toxicology and Environmental Health, Part B 8: 1-37.
Last updated: 2006-01-30 mbl