CICEET Progress Report for the period 9/16/06 Through 3/15/07

Project Title: A fiber optic microarray technology for the detection and enumeration of harmful algal bloom (HAB) species
Principal Investigator(s): Donald M. Anderson and David Walt
Project Start Date: 9/01/05 Project Objectives for This Reporting Period
Objectives
During this reporting period, work focused on the following project aims:
Objective 2: Design and test probes for toxic Pseudo-nitzschia spp. from the Gulf of Maine;
Objective 3: Design and test a second probe pair for each species, to incorporate redundancy into the array;
Objective 5: Refine assay conditions to reduce processing time

Tasks to meet objectives
- synthesize probe sets for Alexandrium fundyense, A. ostenfeldii and Pseudo-nitzschia
- prepare arrays for testing new probe sets
- test RNA extraction protocols for use with micro-fluidic chips

Progress on Tasks
Design and testing of probes During this reporting period we have continued our efforts to expand the fiber-optic array by incorporating probes for Pseudo-nitzschia and adding redundant probe sets for our two Alexandrium species. The incorporation of redundant probe sets for each target should make the measurements more reliable, by reducing the potential for both false negative and false positive detections in environmental samples. A positive or negative signal determination is made only when signal coincidence from several probe types occurs. At present, we have two probe sets each for A. fundyense and A. ostenfeldii, one that targets the large subunit ribosomal region and another that targets the 5.8S rRNA. We have now also designed a probe set to target Pseudo-nitzschia diatoms. Our original fiber optic array used probes targeting west coast Pseudo-nitzschia, which were suitable for proof-of-concept studies using cultured organisms. However, our sequence analyses of Pseudo-nitzschia strains from the northeastern U.S. (representing four different sequence types) show that they do not match any of the existing LSU-targeted probes for Pseudo-nitzschia developed by Chris Scholin of the Monterey Bay Aquarium Research Institute. The strains from the Gulf of Maine show one base pair changes and/or indels in the region of the probe sequences. We have designed a probe for the northeastern strains that targets a portion of the large subunit rRNA and is suitable for detecting Pseudo-nitzschia at the genus level. Additional probes targeting various toxic species is planned when we have completed the toxicity testing of our Pseudo-nitzschia culture collection.

At this time, the probes have been designed and ordered, and we are awaiting delivery. Once the probes arrived, they will be coupled to beads and tested using synthetic targets to ensure that they are working properly. After that, we will proceed to testing with cultured cells and natural samples (see below).

Refining assay conditions We have also been working towards modifying our assay conditions in order to reduce both the overall and the per-sample processing times. In our last report, we described the modification of the hardware components of the fiber optic apparatus to incorporate a microfluidic sample handling system. Prior research in the Walt lab has demonstrated that flowing a target solution across the surface of a sensor array reduces both the limit of detection and required exposure time. During preliminary testing of the polycarbonate microfluidics chips, we realized that our original lysis buffer was causing bubbling in the narrow channels of the chips, impeding proper flow and limiting the effective RNA concentration at the sensor surface. In addition, denaturing agents in the buffer were causing degradation of the chips such that they could not be reused. To mitigate these problems, we investigated the use of RNA extraction and purification methods that would result in a cleaner sample in a relatively inert buffer. We tested four different RNA extraction kits, choosing only those methodologies that would be amenable to automation. The performance of these methods will be compared to our original lysis protocol using the new probe arrays, once they have been validated.

Have the results/data gathered during this reporting period changed the project objectives when compared to your original proposal?
Due to the promising nature of this technology, some new activities have been undertaken that were not anticipated at the time this proposal was written. Specifically, we have shared this technology with Dr. Christopher Scholin, at the Monterey Bay Aquarium Research Institute, who is working to create a fiberoptic module in his environmental sampling processor (ESP) ­ a fully automated remote instrument that can collect water and perform all of the chemistries needed to detect HAB cells. This work was not a project objective explicitly, but is clearly one of the ultimate goals of this research ­ namely to develop a fiberoptic system that can be used in remotely deployed instruments. This work has not detracted from our efforts on other objectives ­ it is entirely a supplemental activity ­ and a very promising one at that.

Dissemination activities during this reporting period
Workshops/ conferences/ trainings coordinated/conducted by your team:
Elements of this project were discussed at an ECOHAB modeling workshop held for another project, but which involves a number of the shellfish managers in the region. During this workshop, we were able to learn a great deal about the specific needs of these managers with respect to types of data, data frequency, locations for sensors, and so forth, that will be useful in guiding the further development of this technology.

Project related presentations/poster sessions at workshops/conference:
D.M. Anderson, 12/06. Multidisciplinary approaches to monitoring, control, and management of harmful algal blooms (HABs). The International Conference on Coastal and Ocean Governance, East Asian Seas Congress 2006, Haikou City, China.

D.M. Anderson, 02/07. The expanding problem of harmful algal blooms: New technologies for monitoring, research and management. The Arabian Seas International Conference on Science and Technology of Aquaculture, Fisheries and Oceanography, Kuwait.

Manuscripts published or submitted for publication:
Anderson, D.M. Multidisciplinary approaches to monitoring, control, and management of harmful algal blooms (HABs). Proceedings of the International Conference on Coastal and Ocean Governance, East Asian Seas Congress 2006 (submitted).

Anderson, D.M. The expanding problem of harmful algal blooms: New technologies for monitoring, research and management. Proceedings of The Arabian Seas International Conference on Science and Technology of Aquaculture, Fisheries and Oceanography (submitted).

Contact with End Users: Feedback was solicited for the current project report.

Student activity (e.g. theses, dissertations, etc.) on the project (please identify students as graduate or undergraduate): Ryan Hayman, a Ph.D. student in the Walt laboratory at Tufts, is working on the microfluidics chips and HAB array system as part of his thesis research. In addition, Lindsay Green, Northeastern University undergraduate student, is working in the Anderson laboratory and is preparing samples for use by the Walt laboratory.

Difficulties
None to report.

Data Generated to date
To date, we have generated sequence and probe data for three species of toxic algae: Alexandrium fundyense, Alexandrium ostenfeldii and Pseudo-nitzschia

Project Objectives for Next Reporting Period

Objectives
During the next reporting period, work will focus on the following project objectives:
Objective 2: design and test probes for toxic Pseudo-nitzschia species from the GoM;
Objective 3: design and test a second probe pair for each species, to incorporate redundancy into the array;
Objective 5: refine hybridization conditions to reduce processing time; and
Objective 6: fully test the 3-species multiplex array using mixed cultures, and simulated and natural field samples.

Work plan to Meet Objectives
The work plan for this next, and last, reporting period focuses on testing the fiber optic array system with the newly designed probe sets, using both cultured and natural samples. First, the new probes will be coupled to beads and tested using synthetic targets. If they are functioning as expected, we will then test the probes individually, against different numbers of cultured target cells. Next, the probes will be combined into a multi-species array, which will be tested against lysates from different combinations of cultured target cells (2-3 species per lysate). The final test will use cultured cells “spiked” into a natural seawater sample, to determine any effects of the complex natural background on the results.

After validating the multiprobe arrays, the last task will be to use the arrays for the analysis of natural samples. We have, in hand, a set of >150 samples collected from the Gulf of Maine in 2005 which contain Alexandrium fundyense and possibly A. ostenfeldii and Pseudo-nitzschia. The A. fundyense cell numbers in these samples have been determined by two other independent methods, making these samples ideal for a comparison of cell detection methodologies. In addition, we will be working with our partners at the Wells NERR in Maine to collect samples from the reserve this spring. These samples will also be analyzed using the fiber optic array system.

During this time we also want to work with our end user and NERR partners to discuss the operational and technical needs for this fiber optic assay. Our efforts will focus on identifying the most effective mechanisms for communicating our project goals and progress to our partners, and the most efficient means of generating feedback from them regarding their needs and desires for cell detection assays and instrumentation.

Dissemination Objectives for next reporting period
As described above, one of our goals is to solicit the involvement of our end users in communicating their needs and desires for instrumentation for HAB monitoring. We also plan to present the test results with the updated fiber optic array system at one or more international meetings (e.g. Pittcon, American Society for Limnology and Oceanography).

Overall Project Timeline Update
There are no changes to the original project timeline.

Expenditures
Expenditures are in the range anticipated for the work accomplished to date.

End User Advisor Feedback
End User Advisor: Michelle Dionne
Organization: Wells National Estuarine Research Reserve
Location: Wells, Maine
Phone number: 207.646.1555x136
E-mail: dionne@wellsnerrcec.lib.me.us

End User Advisor: Darcie A. Couture
Organization: Maine Department of Marine Resources
Location: West Boothbay Harbor, Maine
Phone number: 207.633.9570
E-mail: Darcie.Couture@maine.gov

End User Advisor: Michael Hickey
Organization: Massachusetts Department of Marine Resources
Location: New Bedford, Massachusetts
Phone number: 508.990.2860x122
E-mail: Michael.Hickey@state.ma.us

As with our last report, we have received replies from two of the three end user advisors. We have included the replies of the two end users below, and we will continue to actively solicit feedback from our third end user.

At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
Couture: We continue to view any advances in HAB detection as potentially important to assisting our small agency in protecting public health, while minimizing economic losses to our shellfish industry, especially after the devastating impacts of the 2005 Alexandrium HAB event.

Dionne: [with regard to the application of testing the instrument in the NERR] When combined with our system wide water quality monitoring data, and data from concurrent sampling cruises in the Gulf’s open waters, we should be able to answer questions regarding the origin, movement, concentration and persistence of these species in southern Maine estuarine waters. This information will provide a solid database against which to compare future HAB distribution and abundance patterns in the Reserve’s estuaries. At present, our estuaries do not exhibit symptoms of eutrophication, so our working hypothesis is that HABs enter our estuaries from the Gulf of Maine. However, we will not be surprised if eutrophication becomes an issue for us in the future, given the unbridled growth we are experiencing along Maine’s southwestern coast. Future data such as that generated by your instrument would allow us to determine if and when our estuaries begin to support source populations of these species. Such data could provide a timely rationale for changes in land-use and sewage treatment in our region, in order to restore the ecological state of southcoast Maine estuaries. Beyond our interest in maintaining estuarine ecological functions, we are also interested in providing data useful to the long-term sustainability of harvestable populations of soft-shell clams (Mya arenaria) in the region. The sensors you are developing would certainly be of great use toward that end.

What are the key challenges to application of this technology? Please consider the technology itself as well as issues related to regulation, politics, socio-economic pressures, trends in the field etc.
Couture: The most apparent challenge to the application of this new technology would be the costs associated with the actual use. It is difficult to justify the maintenance of our current funding levels, and would be even more difficult to request additional funding for new technology; our most likely option would be to seek outside funding (grants) to support the use of the technology.

Dionne: none given

Has anything changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?
Couture: At the end of 2006, the state of Maine was granted $2 million in federal relief monies, based on the economic disaster caused by the 2005 Alexandrium HAB event. Based on overwhelming public comments, the Governor determined that the majority of this money would go directly to the shellfish industry as cash relief, while a small portion (~ $390,000) would be used to augment testing and research by the Department of Marine Resources (DMR). This money has been budgeted over the next two years to study the feasibility of commercial depuration of biotoxins, as well as to expand the existing HAB monitoring program from a mostly land-based program, to one that will include near-shore monitoring of sentinel buoys with mussel bags, and phytoplankton samples, by small boat. If we are able to demonstrate a tangible increase in the response to a HAB event with this augmented program, we hope to obtain funding to continue the program beyond two years, and possibly work toward transitioning over completely to a boat-based program in the near future. New technology which would compliment our efforts to work toward a better "warning system", by detecting HABs and HAB toxicity further offshore, rather than already in the shellfish beds, would fit well into the direction in which we hope to see our overall state program going.

Dionne: none given

Questions/comments/ suggestions for the researchers?
Couture: none given

Dionne: We will be very happy to send water samples from the Wells NERR estuaries (Webhannet River and Little River) to test your probes, and learn more about the distribution and abundance of Alexandrium and Pseudo-nitzschia in our system.

PI Response to End User Advisor Feedback
We are pleased that our end users see potential for this technology to assist in their monitoring programs. Of course we, too, are sensitive to the issue of final instrument cost, which is a consideration for research as well as monitoring programs. We will continue our exchange with our end user partners, and are particularly excited about working with Michele Dionne and the staff of the Wells NERR this spring, to collect samples for fiber optic analysis.