Pilot Projects

Overview

M.E. Hahn

The purpose of pilot projects is to assess the feasibility of new areas of study, especially those that are not currently represented in the Woods Hole COHH but would contribute to the overall goals of the COHH program and the Woods Hole center. Pilot projects are intended to support the collection of preliminary data that can be used to generate full proposals to NSF, NIH, or other agencies or organizations. Through this pilot project program, we also hope to:

stimulate collaborative and interdisciplinary research within the center,
recruit scientists not currently involved in the WH-COHH to become participating members of the center and interact with other center investigators,
encourage the use of the WH-COHH genomics core facility, and
oster the application of new technologies and experimental approaches to questions concerning the impact of oceanic processes on public health.

We issued our first annual call for pilot project proposals in June of 2004. The request for proposals was distributed by email to all faculty and research staff at WHOI, MBL, and MIT. Thirteen one-page pre-proposals were received by the deadline of July 15, 2004. These pre-proposals requested a total of $938,557 ($615,230 direct costs). Five were from WHOI, 4 were from MBL, 3 were from MIT, and one was cross-institutional.

Pilot projects were awarded in 2004, 2005, and 2006. For descriptions of the funded projects, please see link at top of page.

These pilot projects are being supported using the funds provided by NIH and NSF as part of the COHH Center grant, supplemented by additional institutional commitment provided by WHOI.

2007 Funded Pilot Projects

Hydrodynamics and transport pathways for fecal microbial populations in a salt marsh and barrier beach system

PI: David Ralston (WHOI, Dept of Applied Ocean Physics and Engineering)

We propose to develop and implement a numerical model of flow and transport in a salt marsh and barrier beach system on Cape Cod, MA. The project will focus on Little Sippewissett Marsh and Wood Neck Beach, a system that during summer months frequently experiences high fecal coliform concentrations that indicate potential for significant impacts on human health and lead to restrictions on recreational use. Potential sources of fecal contamination include failed septic systems, birds, or other animals in the marsh. Recent investigations have found that fecal coliform counts are highly variable temporally (tidally, with precipitation events, and seasonally) and spatially across the marsh and beach. A COHH pilot project currently underway has begun to quantify the spatially heterogeneity of microbial communities in the system, intending to distinguish among different sources of fecal contamination and to develop a broader suite of indicator organisms (Sogin, 2006). The project proposed here will work in conjunction with that microbial community mapping project to quantify transport pathways, residence times, and exchange rates in the marsh. Combining transport mechanisms with the spatially heterogeneous source terms will permit calculation of potential exposure and risk to human health associated with the disparate sources of contamination. The model results will also provide guidance for public health officials to redesign of monitoring efforts to sample at times and locations of maximum potential exposure to elevated coliform concentrations, thereby minimizing human health risks. The tasks in the project include acquiring bathymetric data from existing sources and from new field surveys, constructing a numerical grid from the bathymetry, establishing and collecting data for model boundary conditions, and initial calibration and testing of the model based on available observations in the marsh. The model is intended to serve as a basis for future interdisciplinary studies at the study site – model results will aid in design of field observations, and more extensive field studies will aid in refinement of the model. The salt marsh and barrier beach of the study site represent a common coastal environment, and the impacts on human health of fecal contamination in coastal settings are of concern regionally and nationally. The pilot project will provide a step toward building an integrated program in Little Sippewissett to study linkages among physical, chemical, and biological processes in salt marshes and very shallow estuaries, coastal environments where frequent human interactions make potential health impacts particularly significant.

BMAA, a cyanobacterial neurotoxin, in marine food webs: a pilot project

PI(s): Carl Lamborg, Mak Saito, Paul Drevnick (WHOI, Dept. of Marine Chemistry and Geochemistry)

ß-methylamino-L-alanine (BMAA) is a neurotoxic amino acid produced by cyanobacteria. High concentrations of BMAA in human brain tissue have been linked to neurodegenerative diseases (ALS, Alzheimer’s, Parkinson’s) in Guam and Canada. The source of BMAA in Guam is cyanobacteria in the roots of cycad plants and biomagnification through a unique food web. The source of BMAA in Canada is unknown. A recent study, however, reported that many marine cyanobacteria also produce BMAA. Cyanobacteria are ubiquitous in the ocean and especially abundant in coastal areas that have experienced harmful algal blooms, representing a potentially significant source of BMAA to marine food webs. Fish or shellfish that eat cyanobacteria or otherwise accumulate BMAA may thus pose a health risk to human consumers of seafood. We propose to address the most fundamental question concerning the distribution of BMAA in the temperate coastal ocean: Are BMAA concentrations in seafood high enough to be of concern for human health? We will examine fish and shellfish of commercial, recreational, and subsistence importance for BMAA concentrations. If we find BMAA concentrations that pose a human health risk, (i) this could form the basis of a human health risk assessment for BMAA and (ii) we will have preliminary data to generate a full proposal for further study.

Using signature tagged mutagenesis (STM) to investigate how pandemic Vibrio parahaemolyticus persists in the bacterioplankton and associates with epithelia in the marine environment

PI: Janelle Thompson (MIT, Dept of Civil and Environmental Engineering)

This study will estimate the costs-of-illness associated with human respiratory ailments that arise as the consequence of the aerosolization and coastal to inland transport of brevetoxins from blooms of the marine dinoflagellate, Karenia brevis, in the Gulf of Mexico. The research will develop models to link the occurrence of HAB events in the coastal-ocean with exposures to aerosolized brevetoxins. The researchers will compile datasets and develop models of illness rates that would permit historical estimates of these kinds of impacts and the simulation of future potential impacts. This is a proof-of-concept Pilot Project designed to develop an analytical framework that can be used on a larger scale, using more extensive datasets in the future. It is critical that we understand the costs of natural hazards such as HAB events for at least two reasons. First, the nature of the costs (their effect) and their incidence (who is affected and at what rate) will enable the characterization of feasible actions to mitigate the costs. Second, the scale of the costs will help resource managers, scientists, and the general public to gauge the levels of and need for potential mitigation.

2006 Funded Pilot Projects

Anthropogenic impacts and profiling fecal microbial populations at a salt marsh

PI: Mitch Sogin (MBL)

Fecal coliforms are indicator organisms that warn of possible fecal contamination and its potential impact on human health. Coliform surveillance activities generally rely upon cultivation assays or molecular Microbial Source Tracking (MST) technology to assay for the presence of a particular indicator organism in environmental samples. This project will employ a massively parallel DNA tag sequencing strategy to profile entire microbial communities in the Little Sippewissett Salt Marsh and the barrier Woodneck Beach. This study site is surrounded by ~40 homes, most of which are occupied on a seasonal basis and all are serviced by septic systems. The marsh communicates with Buzzards Bay through a tidal inlet. Multiple times each summer high coliform counts indicate dangerous conditions for recreational use. Possible coliform sources include human waste from failed septic systems, bird populations or other animals. The objective is to use the tag sequence data to locate specific sources of fecal contamination and identify suites of genes that could serve as multi-species indicators of human pollution. The experimental strategy takes advantage of rapidly evolving hypervariable regions in ribosomal RNAs and our ability to generate many thousands of short DNA tag sequences using 454 Life Science’s pyrosequencing sequencing technology on a Roche Genome Sequencer 20 System. Samples will be collected throughout the summer of 2007 and tag sequence will be determined form those microbial populations that were harvested on days corresponding to high coliform count measurements provided by the Barnstable Health Department. Because the technique returns quantitative information about most if not all members of a microbial community, these researchers will be able to track the location of contamination and map its distribution through the marsh.

Transcriptional markers of life cycle transitions in harmful algal blooms

PI: Don Anderson (WHOI )

Bloom dynamics of the red tide dinoflagellate, Alexandrium fundyense are driven in large part by transitions in its life cycle. While these stages are well documented, the biological and oceanographic forces that trigger transitions between the stages are not. A major obstacle to determining the conditions that trigger these transitions is our inability to rapidly identify sexual stage cells. Here, a novel transcriptome experiment will be used to discover molecules that are uniquely expressed by conjugating gamete cells and by germinating cysts. This experiment will utilize sequencing-by-synthesis technology that is newly available through the Bay Paul Center. Data from the experiment will be analyzed in a fashion that is directly analogous to SAGE. Results will be used to leverage a larger, multi-year proposal to verify and validate transcriptional markers discovered through the proposed work.

The economic effects of harmful algal blooms: A pilot project to estimate the costs of human respiratory ailments associated with aerosolized brevetoxins

PI(s): Porter Hoagland and Di Jin (WHOI); Lora Fleming (Miami)

The economics of human health risks from pathogens and toxins in the marine environment

PI(s): Hauke Kite-Powell and Porter Hoagland (WHOI)

These researchers will produce an “order of magnitude” estimate of the annual human health cost imposed on residents of the United States by exposure to pathogens and toxins from the marine environment. The estimate will be derived from a review and synthesis of information in the existing literature on (1) the spatial and temporal prevalence of marine pathogens, (2) the pathways by which they affect humans and the potentially exposed populations, (3) the human health effects of exposure, and (4) the economic cost of resulting medical conditions. The estimate will inform future research on pathogens in the marine environment, allowing scholars and public officials to target pathogens and settings where improved scientific understanding is most likely to produce significant economic benefits, and setting the stage for focused economic analyses. (co-funded with the WHOI Marine Policy Center)

2005 Funded Pilot Projects

Transcriptome profiling in the harmful alga Aureococcus anophagefferens

PI(s): Sonya Dyhrman
Institution(s): WHOI

Aureococcus anophagefferens is a widespread HAB species that has had severe and negative impacts. A. anophagefferens is allelopathic and is thought to produce a suite of natural products including a water soluble neuroactive metabolite, or toxin, that has been implicated in dose-dependent mortality and health decline in model shellfish. The goal of this COHH pilot project is to sequence three Long- SAGE (serial analysis of gene expression) libraries for this organism to examine the A. anophagefferens transcriptome and how it changes with external stressors. The researchers will sequence roughly 30-40,000 tags per library to build a comprehensive view of the transcriptome.

Beach pathogens

PI(s): Steve Elgar, Britt Raubenheimer, & Rebecca Gast
Institution(s): WHOI

Pathogens in coastal sediments pose a serious health risk to users of America’s beaches, but the effects of waves, currents, and changes in beach sediment on pathogen distribution are not understood. We hypothesize that sediments contaminated by pathogens (eg, from sewage) can be exposed when wind, waves, and currents cause changes in the beach configuration (eg, erosion or accretion), potentially creating additional human health hazards, via both direct contact with contaminated sand and exposure to pathogens carried by spray from breaking waves in the surf. To examine how physical forces may impact sewage-associated microbes on beaches, we will investigate the effects of waves, currents, and changes in beach sediment on the quantity and distribution of Legionella spp. Legionella species will be surveyed using PCR amplification before and after an early-fallseason large-wave event (when significant sediment erosion is likely). Legionella in samples of sand from the beach and the surfzone will be correlated with wave conditions and changes in sediment levels (eg, erosion and accretion).

Names-based cyberinformatics tools for rapid response communications and outreach during event management – a pilot based on harmful algal blooms in NE US coastal waters

PI(s): Patterson, David J. (MBL) and Anderson, Don (WHOI)
Institution(s): MBL

Algal blooms are increasing in frequency, extent and significance. The objective of this project is to promote human health by applying new informatics technologies for biology to improve communication among the public and stakeholders in response to a bloom event. The primary deliverable will be a pilot template for a web site that can rapidly call upon expert sources of information, inherit previously known but relevant information, can add local content and will combine the information dynamically in a very flexible environment. At the core of this project lie original internet services that use the names of organisms to discover and manage biological information. Taxonomic indexing is a biologically informed suite of services that uses taxonomic knowledge and awareness of nomenclatural conventions to bring together information that has been cataloged under different names. Around such services we are assembling modular software that allow us to combine distributed and local knowledge in flexible, interoperable, and scaleable web environments called STAR*sites. The pilot site will exploit the 2005 NE US Alexandrium bloom to demonstrate the feasibility of rapidly combining expert information from multiple sources with locally generated data.

2004 Funded Pilot Projects

Characterization of a cyanobacterial anti-algal compound

Eric Webb and Chris Reddy, Woods Hole Oceanographic Institution

Cyanobacteria are well-known, ubiquitous components of the biosphere whose most important role is in the global carbon cycle - where they remove CO₂ from the atmosphere and subsequently produce O₂. Over geologic time, this physiological activity was responsible for producing the oxygenated atmosphere that was required for human evolution and life. In addition to this important biogeochemical role, cyanobacteria have been shown to be potent producers of natural products (antibiotics, toxins, etc).

Recent work from the Webb laboratory has demonstrated that Microcoleus PCC7420, a filamentous, non-diazotrophic cyanobacterium isolated from a Woods Hole salt marsh, contains two gene families that have been implicated in natural product synthesis: non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS). Additional biochemical work has shown that this strain produces a compound(s) that inhibits the growth of other cyanobacteria. Cell-free extracts from Microcoleus PCC7420 consistently inhibited the growth of two test cyanobacterial strains.

Herein we propose to purify and structurally characterize the growth-inhibiting compound and determine the environmental cues that regulate its expression. These data and a partial genome sequence (obtained by the Sogin Laboratory) will be indispensable in identifying the genes responsible for its synthesis. Further experiments will be designed to determine if this compound is a cyanobacterial-specific inhibitory compound, a more general antimicrobial or antibiotic, or a eukaryotic and prokaryotic phytoplankton inhibitor.

Microbial natural products already serve as one of the foundations of pharmacology. Although it has been demonstrated that cyanobacteria produce natural products, due to the perceived difficulty in culturing these organisms, relatively few compounds have been characterized to date. In the future cyanobacterially-derived natural products might be used in diverse sectors of society, including medicine, agriculture, and biocontrol.

We plan to use the data from this study to obtain NSF funding to characterize the ecological significance of this compound in the salt marsh ecosystem and pursue NIH funding to characterize additional activities observed in other strains of cyanobacteria.

Cnidarian toxins against voltage-gated Ca²⁺ channels

Robert Greenberg, Marine Biological Laboratory

Cnidarians such as jellyfish and sea anemones produce venoms that are comprised of a variety of toxins. Several of these toxins have been characterized and are targeted against specific receptors and ion channels in excitable cells. For example, polypeptide sea anemone toxins have dramatic effects on voltage-gated sodium channels. They bind selectively to these channels and inhibit inactivation of the channel.

Peptide toxins from sea anemones have also been shown to selectively inhibit different types of voltage-gated potassium channels. Voltage-gated calcium (Ca²⁺) channels are critical components of excitable cells. They open in response to changes in membrane potential and regulate levels of intracellular Ca²⁺, an important second messenger. Ca²⁺ channels are targets for several agents used to treat cardiovascular diseases, and they are also being exploited as targets for pharmacological management of pain, epilepsy, and neural ischemia and stroke, as well as other conditions.

Ca²⁺ channels are also acted upon by toxins from arthropods and molluscs. Many cnidarian venoms serve to paralyze their prey, and Ca²⁺ channels associated with the neuromuscular junction may be specifically targeted by toxins found within these venoms.

This project will use heterologously expressed Ca²⁺ channels to screen cnidarian venoms for toxins that interact with these channels. Various species of cnidarians will be collected from east-coast waters, and crude venom extracts will be tested for their effects on expressed Ca²⁺ currents. If specific effects are found, the active toxin components will be isolated by standard biochemical approaches (eg, HPLC), and characterized. These experiments may provide new pharmacological tools for analysis of these channels, and may eventually lead to novel drugs for therapeutic use.

Marine phage as vectors of gene transfer between marine bacteria and bacterial pathogens

Jon King, Massachusetts Institute of Technology

Bacterial infections continue to be a major source of disease and mortality worldwide. A diverse set of genes and gene clusters necessary for bacterial pathogenesis have been carefully documented in the last decade. A subset of these virulence genes are found encoded within facultative pathogens by bacteriophages in the integrated, or lysogenic state (prophages). Conversion of avirulent bacteria to virulent forms by phage lysogeny has been demonstrated in the laboratory and inferred through genomic comparisons between closely related bacterial strains differing in pathogenicity. However, very little is known of the sources and environmental distribution of phage-encoded virulence genes or the evolutionary routes through which they have been acquired by their bacterial hosts.

Bacteriophages are extremely abundant in marine waters, with numbers approaching 107/ml. Recent genomic data suggest that phages of diverse hosts can exchange sequences within a global gene pool. This exchange is reflected in the surprising number of genetic and structural features common between phages of marine heterotrophic bacteria, phages of marine photosynthetic bacteria, and well studied phages of enteric bacteria, including the types causing human diseases. Therefore, the ocean may be an important reservoir of phageencoded virulence genes and may be an active site for phage mediated exchange of these sequences between bacterial populations.

The overall goals of this project are to investigate whether phage-borne genes or gene clusters associated with virulence effects in bacterial pathogens of humans, are also found among phages infecting marine photosynthetic bacteria.

The experiments involve:

a) Isolation of phages of marine and enteric bacteria from estuaries and coastal waters where enteric bacteria from terrestrial runoff or sewage effluents mix with coastal ocean waters carrying marine bacteria. b) Using oligonucleotide probes for known phage-borne virulence genes, assaying filtered water samples for these sequences. If detected, further investigation would determine whether they were associated with isolatable phages of either laboratory enteric bacteria and/or coastal ecotypes of the marine obligate photoautoroph, Synechococcus.

Positive results would open the possibility of identifying sources of extant as well as emerging pathogenic sequences through more systematic investigation of marine microorganisms.