MERHAB 2005: Project Summaries
Institution: University of Maine
Institution: Woods Hole Oceanographic Institution
Investigators: Sonya T. Dyhrman and Deana Erdner
Dinoflagellates of the genus Alexandrium can produce a suite of potent neurotoxins that cause paralytic shellfish poisoning (PSP) in humans, and can have serious deleterious impacts on public health and economic resources.Alexandrium and related PSP-toxicity is a problem of global scale. Within this genus, Alexandrium catenella is widespread in the northwestern part of North America, including the Puget Sound, and is responsible for seasonal harmful algal blooms in these regions. Even at low cell densities, A. catenella toxins can accumulate in shellfish and result in PSP. As a result, accurate measurements ofA. catenella distributions, particularly at low cell density, are critical to continued PSP monitoring and mitigation efforts. Towards this end a specific, sensitive, and high throughput real-time quantitative PCR (qPCR) method has recently been developed to assay the abundance of A. catenella . Laboratory validation indicates that the qPCR assay is sensitive enough to detect 10 cells per DNA extraction, and that it is very specific. This specificity is critical for work on harmful algal blooms (HAB), where toxic species are present in mixed communities of non-toxic phytoplankton. The overall goal of this work is to interface a proven, high-sensitivity detection method for A. catenellainto existing PSP monitoring efforts and to examine its efficacy in predicting or serving as an early warning of shellfish PSP toxicity. Specific objectives of the work plan are outlined in the project description, but key elements of the work plan are as follows:
- Participate in high frequency seasonal sampling and qPCR analysis of water column samples from the 40 Sentinel Sites used for PSP testing by the Washington State Department of Health in the Puget Sound.
- Map catenella abundance over two seasonal cycles (April - November) at the 40 different Sentinel Sites.
- Compare catenella water column abundances at different sites with PSP Impact Category and the yearly estimate of PSP Impact Factor as defined by the Washington State Department of Health Office of Food Safety and Shellfish Programs to determine the extent to which qPCR may be used as a method for early warning of a PSP event.
- Use the resulting data set as a teaching and research tool for undergraduates in the Harmful Algae Research Program funded through the NOAA Career 2004 Program.
This research directly relates to the overarching goal of the MERHAB program: to incorporate tools from harmful algal bloom (HAB) research programs into ongoing HAB monitoring programs. Specifically, this work will partner with existing monitoring efforts in the Puget Sound and it will result in a field-validated method for quantifying A. catenella that could benefit monitoring studies in the Puget Sound region and elsewhere. Furthermore, this research would provide a framework with which to teach and prepare the next generation of coastal ocean scientists and managers by partnering with our NOAA-funded career development program.
Institution: Florida Environmental Research Institute
Institution: The University of Texas at Austin
Institutions: NOAA NESDIS, Chesapeake Research Consortium, University of Maryland Center for Environmental Science - Horn Point Lab, University of Illinois, Evansville, Maryland Department of Natural Resources
Institutions: Center for Marine Science-UNCW , Florida Fish and Wildlife Research Institute, Mote Marine Lab
Investigators: J. Naar, D.G. Baden, A. Bourdelais, CJ Wright, K.A. Steidinger, L. Flewelling, R. Pierce
In the Gulf of Mexico, blooms of the toxic dinoflagellate Karenia brevis cause ecological disasters, result in human respiratory distress and contaminated seafood. With support of a previously funded MERHAB project we had completed the development of a new enzyme immunoassay (ELISA) for brevetoxin analysis (1). This assay has been show to be very well-adapted for assessing human exposure to aerosolized toxins (2) (ref), diagnosing brevetoxin poisoning during mass mortalities of marine mammals (3), identifying new brevetoxin producing microalgae (4) as well as identifying some unexpected vectors of brevetoxin to higher trophic levels (3). This versatility is due to: 1) a sensitivity for brevetoxins in the nanomolar range, 2) a specificity for both brevetoxins and brevetoxin metabolites, and 3) a simplicity and absence of elevated matrix effects allowing analyses of both environmental (seawater, sea-spray, and air-filter) and biological (fish, bird and mammalian tissues and body fluids, shellfish extracts and homogenates). Since the early 197Os, the mouse bioassay has been the only FDA-approved method of shellfish monitoring. Because this assay is labor-intensive, requires the use of dangerous solvents and the destruction of many animals, analyses are restricted to very few laboratories with a low through-put. The development of a faster, more efficient technology to replace this assay has long been a goal of regulatory and scientific communities. The ELISA methodology does not require expensive facilities, the use of radioactive materials or dangerous solvents while providing better sensitivity and reducing the time required for analysis. Additionally, the ELISA can be performed on shellfish meat as well as extracts and, using different methods, parent brevetoxins and metabolites can be analyzed together or individually. A preliminary multi-laboratory evaluation (5) has shown that the ELISA appears to be a very good candidate to replace the mouse bioassay. The method was recommended in September of 2004 by the NSP subgroup of the AOAC task force to be evaluated as an alternative method to replace the mouse bioassay. Although members of the NSP subgroup agreed on the analytical method by itself, there is still a lot unknown regarding the toxins implicated in human poisonings, and the toxins that need to be monitored to ensure human safety. The ultimate objectives of this study are to: 1) identify in shellfish species of economical importance what compounds are implicated in the overall shellfish toxicity, 2) define what sample preparation is required to ensure an accurate evaluation of toxicity, 3) evaluate the performance of the ELISA in a selected area where shellfish will be monitored by both regulatory and alternative methods, 4) to prepare standardized material to perform an independent multi-laboratory evaluation of the assay.
- J. Naar, A. Bourdelais, C. Tomas, J. Kubanek, P.L. Whitney, et. Al. (2002) A competitive ELISA to detect brevetoxins from Karenia brevis (formerlyGymnodinium breve ) in seawater, shellfish, and mammalian body fluid. EHP110(2): 179-185.
- Cheng YS, Zhou Y, Irvin CM, Pierces RH, Naar J, Backer LC, Fleming LE, Kirkpatrick B, Baden DG. 2005. Characterization of Marine Aerosol for Assessment of Human Exposure to Brevetoxins. EHP 112:000-000.
- Flewelling L, Naar J, Abbott J, Baden DG. et al. Red tides and marine mammal mortalities, Nature 435: 755-756
- Bourdelais A., Tomas C.R., Naar J., Kubanek J., Baden D.G. (2002) New Fish-Killing Alga in Coastal Delaware Produces Neurotoxins. EHP 110: 465-470
- Dickey RW, Plakas SM, Jester ELE, ElSiad KR, Johannessen JN, et al. (2004) Multi-laboratory study of five methods for the determination of brevetoxins in shellfish tissue extracts. in Harmful Algae 2002 (Steidinger KA, Landsberg JH; Tomas CR and Vargo GA eds) St Petersburg Florida, USA 300:302
Institution: Quinault Indian Nation (QIN)
Institution(s): University of Southern California, University of California, Los Angeles, University of California Santa Cruz, Southern California Water Research Project.