CICEET Progress Report for the period 9/01/05 Through 3/15/06

Project Title: A fiber optic microarray technology for the detection and enumeration of harmful algal bloom (HAB) species
Principal Investigator(s): Donald M. Anderson, David Walt
Project Start Date: September 1, 2005

Tables

Table 1

Tasks to meet objectives
Note that funding was only received in mid October, so the time for project activities has been limited.

Because our initial capture probe for A. ostenfeldii showed a detection limit of ~50 cells, we designed a different capture probe, designated AO2. This capture probe was tested with both synthetic target and A. ostenfeldii cells and was able to detect as few as 5 A. ostenfeldii cells. With this new A. ostenfeldii probe and our existing A. fundyense and P. australis probes, all of our current assays have a detection limit of < 5 cells (Table 1).

Work has also begun towards designing a second probe pair for each species on the array (Objective 3). The incorporation of a second sandwich hybridization probe pair for each target will 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. To this end, we have collected sequence data for a different region of the ribosomal DNA operon (our current probes target the large subunit ribosomal DNA gene) from a number of species, including our target organisms, as well as closely related species and species that may co-occur in the region. We will use this data and specialized software to design a second capture probe for A. fundyense and A. ostenfeldii that will be incorporated into a single probe array for testing. At this point, we are not yet able to expand our Pseudo-nitzschia probe sets. Our current Pseudo-nitzschia probe targets a west coast strain that we have been using as a test organism, as no east coast Pseudo-nitzschia cultures are available. One objective of this project is to gather sequence data for east coast Pseudo-nitzschia species and to design probes specific to local species using that information. That process is now underway (see below).

Work Plan for Next Reporting Period
During the next six months, our efforts will be directed towards three of our objectives:
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; and
Objective 4: Work with coastal managers to determine desired detection limits, precision, and other operational characteristics for the assay and expected instrumentation.

Beginning this spring, we will collect seawater samples from coastal Maine in conjunction with our NERR and end-user partners. These samples should provide a source of live Pseudo-nitzschia cells that we can use to establish cultures. These cultures will then be characterized by sequencing of their ribosomal RNA genes as well as testing for toxicity (domoic acid production). These sequences will be used for probe design and the cultures will be used for probe and array testing.

We will also continue our work to design a second capture probe for each of our target organisms. We expect that by the end of the next reporting period we will have single probe arrays that incorporate our new probe sequences, ready for testing with cultured cells.

The next six months is also a time to work with our end user and NERR partners to discuss the operational and technical instrumentation needs for this fiber optic assay. After we establish our probe sets, we will move to a phase of assay optimization, the goals of which depend upon the input from end users. We will work towards developing the appropriate mechanism for communicating our project goals and progress to our partners, and soliciting feedback from them.

Anticipated Success in Meeting Project Objectives
We are very satisfied with our progress to date. We have completed one project objective and begun a second within this first project period. At this time, we have not run into any difficulties or unexpected issues that would require us to deviate from our current project timeline.

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

Preliminary Data
See Table 1.

Dissemination
Publications: none
Workshops: none
Conferences: none
Manuals, Protocols: none
Outreach Activities: none
Contact With End Users: Feedback was solicited for the current project report. As detailed above, more extensive discussions with end users are planned for next project period.
Patent, Copyright, Invention Disclosure Activity: none

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

End User Advisor Feedback
Name: J. Michael Hickey
Organization: Massachusetts Division of Marine Fisheries
Location: 50A Portside Drive, Pocasset, MA 02559
Phone number: (508) 563-1779, x122
E-mail: Michael.Hickey@state.ma.us

1) At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
Technology (probes) that will accurately detect and enumerate algal cells responsible for harmful algal blooms would be a boon to coastal managers tasked with protecting public health especially if the information was available in real time. This would provide early warning of an impending or actual event and would allow managers to direct resources to those areas where cell counts were high or rising to conduct shellfish sampling to determine toxicity in the case of PSP, ASP or DSP. In addition cell identification and counts coupled with oceanographic information would help to understand HAB bloom dynamics and transport.

2) What, if anything, has changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?
Nothing has changed the potential applicability of this project.

3) Do you see any key challenges that the researchers may want to address or keep in mind?
No.

4) Does this report offer you enough information to adequately address the above questions?
Yes.

5) Other feedback?
It appears that good progress is being made.

Name: Michele Dionne
Organization: Wells National Estuarine Research Reserve
Location: 342 Laudholm Farm Road, Wells, Maine 04090
Phone number: 207-646-1555x136
E-mail: dionne@wellsnerrcec.lib.me.us

1) At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
Probes to monitor HAB dynamics would be an important biological addition to the NERRS system-wide monitoring program, especially if we add real-time telemetry to the probes, as we are now doing for physico-chemical and fluorometric parameters we currently measure with the YSI 6600 instruments. If these probes were deployed in parallel with probes on IOOS buoys, we could provide an estuarine end-member to the sort of data set that is needed to understand the drivers and dynamics of HAB populations.

2) What, if anything, has changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?
Nothing has changed the potential applicability of this project.

3) Do you see any key challenges that the researchers may want to address or keep in mind?
I have nothing to add at this juncture.

4) Does this report offer you enough information to adequately address the above questions?
Yes.

Name: Darcie Couture
Organization: Maine Dept. of Marine Resources
Location: W. Boothbay Harbor, Maine
Phone number: (207) 633-9570
E-mail: darcie.couture@maine.gov

1) At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
As a state agency, our Department is often faced with unstable budget and staff issues as administrations and legislatures change; it is therefore vital for us to develop a program which can accommodate these uncertainties, yet also function effectively as a Public Health Division program. Our current strategy for managing biotoxins in our shellfish growing areas is very labor intensive, and new technologies such as these probes would give us a tool that is both easy to use and accurate for predicting early levels of HABs. The information that these probes could provide would be extremely important in giving us an early warning when toxic species are beginning to appear in an area, allowing us to focus our monitoring resources more effectively.

2) What, if anything, has changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?
N/A.

3) Do you see any key challenges that the researchers may want to address or keep in mind?
As the researchers move on to looking for Pseudo-nitzschia species in the Gulf of Maine, close coordination between Maine phytoplankton monitors and the researchers will be very important in order to catch the blooms and collect viable specimens.

4) Does this report offer you enough information to adequately address the above questions?
This report, in conjunction with ongoing communications with the researchers, is adequate.

5) Other feedback?
None at this time.