a p r 2001 september 1, 2000 through august 31,...

Connecticut Delaware Maine Maryland Massachusetts New Hampshire New Jersey New York Pennsylvania Rhode Island Vermont Washington, D.C. West Virginia

ANNUAL PROGRESS REPORT 2001 September 1, 2000 through August 31, 2001

Northeastern Regional Aquaculture Center Annual Report 9/1/00 – 8/31/01

Compiled by the Northeastern Regional Aquaculture Staff:

Julie Smith, Executive Assistant Karen E. Tavares, Administrative Services

Andrew Kant, Office Assistant

Work summarized in this report was supported in part by Grant No’s. 96-38500-3032, 97-38500-4641, 98-38500-5917 from the Cooperative State Research, Education, and Extension Service, sponsored by

the United States Department of Agriculture.

___________________________

The University of Massachusetts Dartmouth serves as the host institute for NRAC. UMass Dartmouth is an Equal Opportunity and Affirmative Action Employer.

MISSION

NRAC is a principal public forum for the advancement and dissemination of science and technology needed by Northeastern aquacultural producers and support industries. NRAC facilitates regional stakeholder communications—linking industry and government representatives to university scientists and educators—guiding and stimulating regional research and outreach initiatives. NRAC focuses on science and education that will have a direct impact on attaining long-term public benefits through enhanced aquacultural development in the region. NRAC-sponsored projects emphasize science and education to stimulate growth of the industry—measured in size and numbers of aquacultural enterprises—through development and dissemination of profitable and environmentally benign technologies.

NRAC serves the following 12 states, and Washington, D.C.:

CT, DE, ME, MD, MA, NH, NJ, NY, PA, RI, VT, WV

Northeastern Regional Aquaculture Center i Annual Report 9/1/00 – 8/31/01

TABLE OF CONTENTS I. Introduction ..........................................................................................................................................................1

II. Organization and Administration..........................................................................................................................1

A. Board of Directors .........................................................................................................................................2

B. TIAC..............................................................................................................................................................3

C. State Aquaculture (Extension Contacts, Associations, Coordinators) ...........................................................4

III. Project Status ........................................................................................................................................................9

A. Completed Projects .........................................................................................................................9 B. Ongoing Projects .............................................................................................................................9 C. New Projects ...................................................................................................................................9 D. Project Development during this Period..........................................................................................9 E. Project Development for 2002.........................................................................................................9 F. List of Completed and Ongoing Projects ...................................................................................... 10

IV. Project Completion Reports

97-2 “Expansion of the ‘Fishguts’ Fish Anatomy, Health and Necropsy Software Training Program for the Aquaculture Community” ............................................................................. .12 97-7 “Developing a Sustainable Market for Northeast U.S.A. Aquacultured Products by Effecting Attitudinal Changes in the Foodservice/Restaurant Sector ........................................................18 98-4 “Determination of Optimal Swimbladder Inflation in Striped Bass Larvae Reared in Intensive

Systems” ....................................................................................................................................36 98-6 “Development and Application of Multiplex PCR for Screening of Shellfish Pathogens”.............40

V. Project Progress Reports

98-5 “Improving Intestinal and Renal Phosphate Absorption in Fish”....................................................46 98-7 “Aquaculture Curricula Resource Guide Publication and Distribution” .........................................54 00-2 “Improving Larval Survival for Black Sea Bass Aquaculture”.......................................................57 00-3 “Surveillance of Infectious Anemia Virus (ISAV) in the Northeast”..............................................59 00-4 “Development of a Producer’s Practical Guide to Intensive Aquaculture”.....................................62 00-5 “An Industry Directed Feasibility Study of the Razor Clam (Ensis directus) as a Candidate for

Intertidal and Shallow Subtidal Culture in the Northeastern U.S.”............................................66 VI. Project Support Information .................................................................................................................................69

Northeastern Regional Aquaculture Center 1 Annual Report 9/1/00 – 8/31/01

I. INTRODUCTION The Northeastern Regional Aquaculture Center (NRAC) is one of five Regional Aquaculture Centers which were established and authorized by the U.S. Congress under Title XIV of the Agriculture and Food Act of 1980 and the Food Security Act of 1985. In addition to the northeastern region, aquaculture centers have been established in the north-central, southern and western regions of the continental United States and in Hawaii. The Regional Aquaculture Centers (RACs) are administered by the United States Department of Agriculture (USDA) which was given Federal leadership in aquaculture by Congress through the National Aquaculture Act of 1980.

USDA leadership is assigned to the Office of Aquaculture which is located in the Cooperative State Research, Education and Extension Service (CSREES) of USDA. The RACs are administrative agencies which encourage and fund cooperative and collaborative aquaculture research and extension educational programs that have regional and/or national application. The RACs are organized to take advantage of the best aquaculture science, educational skills, facilities and extension services in the United States; all projects are driven by industry needs and are designed to directly impact commercial development of aquaculture in all states and U.S. territories.

II. ORGANIZATION AND ADMINISTRATION

NRAC encompasses 12 states (Connecticut, Delaware, Maine, Maryland, Massachusetts, New Hampshire, New Jersey, New York, Pennsylvania, Rhode Island, Vermont and West Virginia) and the District of Columbia. The NRAC administrative office is located on the campus of the University of Massachusetts Dartmouth, in North Dartmouth, MA. The office is staffed by an Executive Director, Executive Assistant, Administrative Assistant (all three are full-time positions) and part-time student aides. The Center is responsible for coordinating proposals and managing grants, serving as fiscal agent for administering grant monies, arranging external peer reviews of proposals, maintaining liaison with other Regional Aquaculture Centers and USDA officials, and for other administrative matters as required in the daily management of the research grants. A Board of Directors (Board), representing the region's aquaculture industries, academic institutions and government agencies, establishes policy and provides overall direction for NRAC. The Board elects from itself a 6-member Executive Committee which oversees matters of operating procedure for NRAC. A Memorandum of Understanding (MOU), enacted in 1988, describes and governs the relationships between member institutions in NRAC. Research and extension priorities are established by a 24-member Technical/Industry Advisory Council (TIAC) in consultation with the aquaculture industry. The TIAC has a broad regional makeup and represents scientists, extension agents and industry

members, with varied aquaculture expertise. The TIAC consists of a 12-member Technical Committee (TC) and a 12-member Industry Committee (IC). Nominations for the TIAC are solicited widely throughout the Northeast region. During this report period, recruitment efforts to secure a permanent Executive Director took place. Dr. Tomas Jamir was hired on August 8, 2001. Mr. William Wise of the Marine Sciences Research Center, State University of New York at Stony Brook served as Chairman of NRAC’s Board of Directors. Mr. Wilson Sallum of Eastern Fish Farms, Inc., of Tiverton, Rhode Island, and Dr. Dale Leavitt of the SouthEastern Massachusetts Aquaculture Center located at the Massachusetts Maritime Academy in Barnstable County, Massachusetts served as Industry Committee and Technical Committee Co-Chairs of the TIAC, respectively.


Board of Directors Dr. Acacia Alcivar-Warren Tufts University School of Veterinary Medicine 200 Westboro Rd. North Grafton, MA 01536 Dr. Bruce Barber University of Maine School of Marine Sciences 5735 Hitchner Hall Orono, ME 04469-5735 *Dr. Lavon L. Bartel Northeast Extension Director’s Designee University of Maine Cooperative Extension 5741 Libby Hall - Rm 102 Orono, ME 04469-5741 *Mr. Kenneth L. Bergstrom Massachusetts Aquaculture Association 237 North Valley Road Pelham, MA 01002-9770 Dr. Donald F. Boesch President, CEES University of Maryland System P.O. Box 775 Cambridge, MD 21613 Dr. Carolyn B. Brooks Dean, School of Agricultural & Natural Sciences Executive Assistant to President 1890 Research Director University of Maryland Eastern Shore Back Bone Rd - JT Williams Bldg. Princess Anne, MD 21853-1299 Dr. Richard T. Burke Associate Vice Chancellor Grants, Contracts and Sponsored Programs University of Massachusetts-Dartmouth Administration 011 285 Old Westport Rd. North Dartmouth, MA 02747 *Mr. Walter Canzonier New Jersey Aquaculture Association P.O. Box 662 Port Norris, NJ 08349

Dr. Richard (Dick) A. Cooper Director, Marine Sciences Institute University of Connecticut Avery Point Campus Groton, CT 06340 Dr. Patrick (Pat) Gaffney College of Marine Studies University of Delaware 700 Pilottown Rd. Lewes, DE 19958-1298 Mr. Jeffrey Gardner Ocean State (RI) Aquaculture Association Shellfish for You 227 Shore Road Westerly, RI 02891 Mr. Clifford A. Goudey Marine Advisory Leader MIT Sea Grant College Program Bldg. NE 20-376 3 Cambridge Center Cambridge, MA 02139 *Mr. Michael Hastings Maine Aquaculture Association Maine Aquaculture Innovation Center 5717 Corbett Hall, Room 438 Orono, ME 04469-5717 Dr. Jurij Homziak Lake Champlain Sea Grant Program School of Natural Resources University of Vermont 317 Aiken Center Burlington, VT 05405-0088 *Dr. Alan M. Kuzirian Associate Scientist 7 MBL Woods Hole, MA 02543 Dr. William (Bill) C. McComb Dept. of Forestry & Wildlife Management Holdsworth Natural Resource Center University of Massachusetts Amherst, MA 01003-4210


Dr. Judith (Judy) McDowell Woods Hole Sea Grant Woods Hole Oceanographic Institution 193 Oyster Pond Rd., CRL 213 Woods Hole, MA 02543-1525 Mr. Andrew Melick Pennsylvania Aquaculture Association Melick Aquafeed, Inc. 139 South First St. Catawissa, PA 17820 Mr. David Morehouse New York Aquaculture Association Morehouse Bait Farm 3435 Seybolt Rd. Seneca Falls, NY 13148 Mr. Melvin Murrel New Hampshire Aquaculture Association R1 Fisher Road Box 162-B Deering, NH 03244 Mr. Rob Nichols West Virginia Aquaculture Association West Virginia Department of Agriculture Marketing & Development Division 1900 Kanawha Blvd. East Charleston, WV 25305-0178 Dr. Larry A. Nielsen Director, School of Forest Resources The Pennsylvania State University 113 Ferguson Building University Park, PA 16802 Dr. Eric Powell Director, Haskin Shellfish Laboratory Rutgers University 6959 Miller Avenue Port Norris, NJ 08349 Dr. Andrew A. Rosenberg Dean of Life Sciences & Agriculture University of New Hampshire 59 College Rd. Taylor Hall Durham, NH 03824 Mr. William Wise Director, Living Marine Resources Inst. SUNY at Stony Brook Stony Brook, NY 11794-5000

**Dr. Gloria Wyche-Moore Director, Agricultural Experiment Station, MB4404, University of DC 4200 Connecticut Ave. NW, Bldg. 52, Rm. 322-D Washington, DC 20008

* Executive Committee **Board Chair

TIAC

Industry Mr. William Bason Delmarva Aquatics 256 Brick Store Landing Road Smyrna, DE 19977 Ms. Deborah J. Gile Hillsborough Trout Farm 186 Old Henniker Road Hillsboro, NH 03244 Jurica Jug-Dujakovic Chief Technology Officer Atlantis Aquaculture Group, Inc. 840 Broad Street Emamanus, PA 18049 Mr. Scott Lindell Fins Technology, LLC. 15 Industrial Blvd. Turners Falls, MA 01376 Mr. Michael Patrick O’Malley Community Economic Development Center of Southeastern Massachusetts 311 Cummington Street New Bedford, MA 02745 Mr. Wilson S. Sallum Eastern Fish Farms Inc. 142 Kenyon Rd. P. O. Box 333 Tiverton, RI 02878 Mr. Curtis Sjolander Mountain Foot Farm 154 Blakely Rd. Wheelok, VT 05851


Mr. Steven D. VanGorder Fresh-Culture Systems, Inc. 630 Independent Road Breinigsville, PA 18031 Mr. Robert Wallace Billingsgate Shellfish & Bait P.O. Box 454 25 Whiffletree Road South Wellsfleet, MA 02663 Mr. Loy Wilkinson Coastal Bio Marine 250 Northrup Street P.O. Box 6 Bridgewater, CT 06752 Technical Ms. Julie Delabbio 219 Rock Street P. O. Box 286 Bluefield, WV 24701 Dr. Dale Honeyfield United States Geological Survey RD #4 Box 63 Wellsboro, PA 16901 Dr. Hauke Kite-Powell Marine Policy Center, MS 41 WHOI Woods Hole, MA 02543-1138 Dr. Dale Leavitt SEMAC c/o Hurley Library Mass Maritime Academy 101 Academy Drive Buzzards Bay, MA 02532 Mr. Jon Lewis Maine Department of Marine Resources P.O. Box 8 West Boothbay Harbor, ME 04575 Mr. Dana Morse Extension Associate Maine Sea Grant Program UMaine Cooperative Extension Darling Marine Center 193 Clark’s Cove Road Walpole, ME 04573

Ms. Joyce Newman Extension Specialist - Aquaculture University of NH Cooperative Extension Kingman Farm Durham, NH 03824 Dr. Robert A. Robertson University of New Hampshire Dept. of Resource Economics & Development 317 James Hall 56 College Road Durham, New Hampshire 03824 Dr. Kenneth Semmens West Virginia University 1052 Agriculture Science Building P.O. Box 6108 Morgantown, WV 26505-6108 Dr. Inke M. Sunila State of Connecticut Department of Agriculture Bureau of Aquaculture 190 Rogers Avenue P.O. Box 97 Milford, CT 06460 State Aquaculture Extension Contacts CONNECTICUT Ms. Tessa Simlick Connecticut Sea Grant College Program Sea Grant Extension Program 1084 Shennecossett Rd. Groton, CT 06340 Dr. Lance Stewart New London Cooperative Extension 562 New London Turnpike Norwich, CT 06360 DELAWARE Dr. William Daniels Research Assistant Professor/ Extension Specialist, Aquaculture Dept. of Agriculture & Natural Resources Delaware State University 1200 N. Dupont Highway Dover, DE 19901-2277


Mr. John W. Ewart Delaware Aquaculture Resource Center DE Sea Grant Marine Advisory Service College of Marine Studies University of Delaware 700 Pilottown Road Lewes, DE 19958-1298 MAINE Mr. Paul Anderson Cooperative Extension Maine Sea Grant College Program 14 Coburn Hall, University of Maine Orono, ME 04469 Mr. Christopher Bartlett Maine Sea Grant 16 Deep Cove Road Eastport, ME 04631 Mr. Dana L. Morse Extension Associate Maine Sea Grant Program UMaine Cooperative Extension Darling Marine Center 193 Clark’s Cove Road Walpole, ME 04573 Dr. Michael H. Opitz University of Maine Cooperative Extension Dept. of Animal, Veterinary & Aquatic Sciences 5735 Hitchner Hall Orono, ME 04469 MARYLAND Dr. Reginal M. Harrell Regional Extension Director, Region III Maryland Cooperative Extension P.O. Box 169 Queenstown, MD 21658 Dr. Steven Hughes Maryland Cooperative Research Unit University of Maryland Eastern Shore 1116 Trigg Hall Princess Anne, MD 21853 Dr. Donald Meritt Sea Grant Aquaculture Specialist Horn Point Environmental Lab P.O. Box 775 Cambridge, MD 21613

Mr. Tom Rippen Sea Grant Seafood Technology Specialist 30921 Martin Court University of Maryland Eastern Shore Princess Anne, MD 21853 Ms. Jackie Takacs Maryland Sea Grant Marine Agent Chesapeake Biological Lab P.O. Box 38, One Williams Street Solomons, MD 20688-0038 Dr. Daniel E. Terlizzi Center for Marine Biotechnology Columbus Center Suite 236 701 E. Pratt St. Baltimore, MD 21202 Mr. Donald W. Webster Sea Grant Marine Agent UMD, Wye Research & Education Center P.O. Box 169 Queenstown, MD 21658 MASSACHUSETTS Mr. William Burt Barnstable County Extension Service Deeds & Probate Building P.O. Box 367 Barnstable, MA 02630 Dr. Dale Leavitt SEMAC c/o Hurley Library Mass Maritime Academy 101 Academy Drive Buzzards Bay, MA 02532 NEW HAMPSHIRE Mr. Roland Barnaby Extension Educator UNH Cooperative Extension 113 North Rd. Brentwood, NH 03862 *Ms. Joyce Newman Extension Specialist-Aquaculture Univ. of NH Coop. Extension Kingman Farm Durham, NH 03824


NEW JERSEY Mr. George E. Flimlin Extension Center Rutgers Cooperative Extension/NJSGMAS 1623 Whitesville Road Toms River, NJ 08755 NEW YORK *Dr. Michael Timmons Agricultural & Biological Engineering 302 Riley-Robb Hall, Cornell University Ithaca, NY 14853 Mr. Gregg Rivara Cornell Cooperative Extension 3690 Cedar Beach Road Southold, NY 11971 Mr. Christopher F. Smith Cornell Cooperative Extension Marine Education Center 39 Sound Ave. Riverhead, NY 11901 PENNSYLVANIA Mr. Joseph McMullen Penn State Cooperative Extension HC01 Box 13 Spruce Creek, PA 16683 RHODE ISLAND Dr. Michael Rice Dept. of Fisheries, Animal & Vet. Sciences University of Rhode Island Fisheries Center Woodward Hall Rm 23 Kingston, RI 02881 VERMONT Dr. Jurij Homziak Lake Champlain Sea Grant Program School of Natural Resources University of Vermont 317 Aiken Center Burlington, VT 05405-0088

WEST VIRGINIA Dr. Kenneth Semmens West Virginia University 1052 Agriculture Science Building P.O. Box 6108 Morgantown, WV 26505-6108 State Aquaculture Associations Connecticut Aquaculture Association Connecticut Aquaculturist Trade Association 1 Ash Lane Southbury, CT 06488 Delaware Aquaculture Association c/o Delmarva Aquatics P.O. Box 349 Odessa, DE 19730 Maine Aquaculture Association P. O. Box 148 Hallowell, ME 04412 Maryland Aquaculture Association c/o Maryland Coop. Extension University of Maryland Eastern Shore Princess Anne, MD 21853 Massachusetts Aquaculture Association - I c/o 142 Cherry Lane Amherst, MA 01002 Massachusetts Aquaculture Association - II c/o Billingsgate Shellfish & Bait P.O. Box 454 25 Whiffle Tree Road South Wellfleet, MA 02663

New Hampshire Aquaculture Association c/o Sumner Brook Fish Farm 277 Route 16 Ossipee, NH 03864 New Jersey Aquaculture Association c/o Ocean County Extension Center 1623 Whitesville Rd. Toms River, NJ 08755


New York State Aquaculture Association c/o Cold Spring Harbor Fish Hatchery & Aquarium P.O. Box 535, Route 25 A Cold Spring Harbor, NY 11724-0029 Pennsylvania Aquaculture Association c/o Perdue Specialty Feeds 139 S. First St. Catawissa, PA 17820 RI/Ocean State Aquaculture Association [OSAA] c/o Spatco, Ltd P.O.Box 2031 Kingston, RI 02881 [courier - c/o Dr. R. Rheault: 1121 Mooresfield Rd.,Wakefield, RI 02879] Vermont Aquaculture Association c/o Culture Tools P.O. Box 132 Monkton, VT 05469 [courier: c/o Roger Wallace 1269 Bristol Rd., Bristol, VT 05443] West Virginia Aquaculture Association Marketing & Development Division 1900 Kanawha Blvd. East Charleston, WV 25305-0178 State Aquaculture Coordinators CONNECTICUT Mr. John H. Volk Director, Bureau of Aquaculture Department of Agriculture P.O. Box 97 190 Rogers Avenue Milford, CT 06460 DELAWARE Dr. Wesley Towers State Veterinarian Delaware Department of Agriculture 2320 South DuPont Highway Dover, DE 19901-5515 MAINE Vacant

MARYLAND Mr. Roy Castles Director, Aquaculture/Seafood Program MD Dept of Agriculture 50 Harry S. Truman Parkway Annapolis, MD 21401 MASSACHUSETTS Mr. Scott J. Soares Commonwealth of Massachusetts Aquaculture Coordinator Dept. of Food and Agriculture Suite 500 251 Causeway Street Boston, MA 02114-2151 NEW HAMPSHIRE Victoria Sokul Smith Aquaculture Coordinator Department of Agriculture P.O. Box 2042 25 Capitol Street Concord, NH 03302-2042 NEW JERSEY Ms. Linda O’Dierno Fish and Seafood Development Department of Agriculture CN 330, John Fitch Plaza Trenton, NJ 08625 NEW YORK none PENNSYLVANIA Mr. Leo L. Dunn Aquaculture Coordinator Pennsylvania Department of Agriculture 2301 N. Cameron Street - Room 311 Harrisburg, PA 17110-9408 RHODE ISLAND Mr. David Alves Aquaculture Coordinator of Rhode Island Oliver Stedman Government Center 4808 Tower Hill Road Wakefield, RI 02879


VERMONT Ms. Denise Russo Marketing Specialist Department of Agriculture 116 State Street Drawer 20 Montpelier VT 05620-290 WEST VIRGINIA Mr. Robert Williams Director of Marketing Marketing & Development Division W.V. Department of Agriculture 1900 Kanawha Blvd. East Charleston


III. PROJECT STATUS

The following summarizes the status of NRAC’s funded research program during the report period. Information about the financial support levels of active projects is found in Section VI.

A. Completed Projects During the report period four NRAC funded projects were completed. Completion reports are found in Section IV. B. Ongoing Projects Six ongoing projects continued in the report period. Progress reports are found in Section V. C. New Projects One new project was started during the report period.

D. Project Development During the report period, a Request for Proposal (RFP) process was the principal mechanism used to develop NRAC's Annual Plan of Work. Forty-four (44) preproposals were received in response to the RFP for NRAC’s 13th Plan of Work. From these, eighteen (18) full proposals were solicited; fifteen (15) full proposals were eventually received. Of these submitted proposals, the TIAC recommended nine (9) to the Board’s Executive Committee for funding. Seven (7) proposals were approved by the Executive Committee for submission to USDA. E. Project Development for 2001 Funding The Request for Proposals (RFP) process and the Work Group Process will be used to develop the 2001 NRAC Plan of Work.


LIST OF COMPLETED AND ONGOING PROJECTS

PROJECT CODE PROJECT TITLE DURATION FUNDING

LEVEL GRANT

NUMBER

97-2

“Expansion of the “FishGuts” Fish Anatomy, Health and Necropsy Software

Training Program for the Aquaculture Community

6/1/97 – 6/30/01 * Project Completed

52,129.00 19,362.00 71,491.00

96-38500-3032

97-7

“Developing a Sustainable Market for Northeast USA Aqaucultured Products by Effecting Attitudinal Changes in the Food

Service/Restaurant Sector”


31,050.00 15,100.00 46,150.00

96-38500-3032

98-4

“Determination of Optimal Swimbladder Inflation in Striped Bass Larvae Reared in

Intensive Systems”


56,053.00 28,589.00 84,642.00

96-38500-3032 95-38500-1423

98-5

“Improving Intestinal and Renal Phosphate Absorption in Fish” 9/1/98 – 2/28/02

135,850.00 43,530.00 179,380.00

97-38500-4641 95-38500-1423

98-6

“Development and Application of Multiplex PCR for Screening of Shellfish

Pathogens”


64,719.00 77,384.00 142,103.00

97-38500-4641

98-7

“Aquaculture Curricula Resource Guide Publication & Distribution” 2/1/99 – 9/28/01 10,000.00

10,000.00 97-38500-4641

00-2

“Improving Larval Survival for Black Sea Bass Aquaculture” 4/1/99 – 3/1/02

67,855.00 64,571.00 132,426.00

97-38500-4641 98-38500-5917

00-3

“Surveillance of Infectious Anemia Virus (ISAV) In the Northeast” 8/01/00 –7/31/03

49,950.00 44,350.00 44,350.00 134,650.00

98-38500-5917

00-4

“Development of a Producer’s Practical Guide to Intensive Aquaculture” 8/01/00 – 7/31/03 19,878.00

19,878.00 97-38500-4641

00-5

“An Industry Directed Feasilbility Study of the Razor Clam (Ensis directus) as a Candidate for Intertidal and Shallow

Subtidal Culture in the Northeastern U.S.”

01/01/01 – 12/31/02 37,450.00 39,450.00 76,900.00

97-38500-4641 98-38500-5917


IV. PROJECT COMPLETION REPORTS


PROJECT COMPLETION REPORT

97-2 “Expansion of the ‘FishGuts’ Fish Anatomy, Health and Nexcropsy Software Training Program for the Aquaculture Community“

Termination Report Period: July 1, 1997 – August 30, 2001 NRAC Total Funding: $ 71,491 (July 1, 1997 – June 30, 1999) (No-Cost Ext. through August 30, 2000; 2nd through August 30, 2001)

Principal Investigator: Dr. Andrew Kane, University of Maryland, College Park Participating Investigators/ Cooperative Agencies:

Ana Baya Maryland Department of Agriculture Maryland Paul Bowser Cornell University New York Charles Conklin Pennsylvania Aquaculture Association Pennsylvania Robert Durborow Kentucky State University Kentucky John Ewart University of Delaware Delaware

John Harshbarger George Washington University Washington DC Frank Hetrick Maryland Department of Agriculture Virginia Tom Hopkins Maryland Aquaculture Association Maryland Steven Hughes University of Maryland Eastern Shore Maryland Ray Klinger Hunting Creek Fisheries, Inc. Maryland Mike Opitz University of Maine Maine Sarah Poynton The John Hopkins University Maryland Renate Reimschuessel US FDA Center for Vet Medicine Maryland Wolfgang Vogelbein Virginia Institute of Marine Sciences Virginia

Brent Whitaker National Aquarium in Baltimore Maryland REASON FOR TERMINATION: Indicate objective(s) completed, funds terminated, or other specific reason for project termination. End of funded project period. PROJECT OBJECTIVES: List objectives as written in approved proposal. The objectives for this project involve the expansion and revision of the “FishGuts” fish anatomy, health and necropsy software (version 1.0). The proposed scope of work included: 1. The addition of an "aquaculture-friendly"

introductory section, 2. The addition of four new fish species (cultured

species within the NRAC region) to the Species Reports section,

3. A picture atlas ("What's My Lesion" section) showing grossly visible parasites/lesions common in cultured finfish, and

4. A directory of fish pathologists and diagnostic

services available within the NRAC region. The revised program is in final stages of development, and being made cross-platform compatible (playable on Macintosh and Window-based computers), for distribution (as version 2.0) to extension contacts and aquaculture offices throughout the NRAC region. A workshop and a mailed fact sheet will acquaint NRAC contacts with the program. The specific objectives for this project included: Objective 1. Storyboard construction. A storyboard was developed to direct how the media elements (text, pictures, animation, narration, sound, and movies) will be incorporated into the updated FishGuts software program.


Objective 2. New media archival. Media elements for new sections of FishGuts were archived and digitally optimized for use in the interactive software. Objective 3. Software development. Digitally-optimized media elements are being “authored” into an integrated, user-navigable format. This objective will produce a final test version of the new program, ready for final review, revision and debugging. Objective 4. Review of program test version. Collaborating participants will review the final test version of the new program for content and continuity. Objective 5. Progress report to NRAC. Yearly interim reports to NRAC have indicated progress to date and comments regarding the program’s development. Objective 6. Final programming. The final test version of the FishGuts software will be updated to incorporate final media additions, comments from participants (content/continuity) and be debugged for programming errors. The program will be tested on a spectrum of different personal computers to insure compatibility. Objective 7. Mastering. A final version of the software will be made into a hybrid (cross-platform) program (i.e., usable on both Macintosh and Windows-based computers). The hybrid software will be tested on both platforms, further debugged if necessary, “cut” onto a master hybrid disk, and duplicated for distribution. Objective 8. Outreach. Instructional package materials will be developed to accompany the software product. FishGuts will be distributed to all extension contacts and aquaculture offices within the NRAC region through an organized workshop. Additional outreach will include presentations and publication in appropriate aquaculture/scientific venues. Objective 9. Final report to NRAC. This final report, detailing all efforts, has been sent to NRAC describing work accomplished to date, and reasons for delay in final product development and distribution.

ANTICIPATED BENEFITS: State how the project will benefit the aquaculture industry either directly or indirectly. There is an increased need for fish culturists, extension agents, veterinarians, and graduate and veterinary students to care for and prepare diagnostic samples from cultured, wild and feral fish for disease analysis. The updated FishGuts software will provide a valuable teaching tool to assist aquaculturists and technicians with the necessary observation skills and preparative techniques to generate samples for analysis by a trained fish diagnostician, pathologist, or other fish health personnel. Production of the updated FishGuts software will provide a cutting-edge resource to support early disease detection, and will promote the application of appropriate and consistent necropsy (and other preparative) techniques. PRINCIPAL ACCOMPLISHMENTS: Summarize in a concise form the findings for each objective for the duration of the project. Measurement data are to be given in metric units. However, to minimize confusion, a dual system of measurement may be used to express results. A storyboard and over 200 MB of media have been amassed to produce the updated FishGuts software. Media elements include pictures, movies, text, narration and animations that all fit within the existing FishGuts screen layouts and overall design. The scope of work on this project has been expanded thanks to new, outside support from the Maryland Agricultural Extension Service, University of Maryland College of Agriculture and Natural Resources. This funding will support an additional section on “Bacteriology” (beyond the original scope of the NRAC-funded project. The “Bacteriology” section is being co-authored with Dr. Ana Baya, Aquatic Microbiologist. In addition, the section on lesions, entitled “What’s My Lesion,” that was to include external, grossly visable lesions and parasites has been divided into two separate sections: “What’s My Lesion” and “What’s My Parasite.” Media elements have been digitally archived and are being incorporated into the interactive software. Project delays: At least three factors have cumulatively lead to a significant delay in producing the final product within the initial timeframe. These


factors include: (1) The move of the University of Maryland Aquatic Pathobiology Center (APC) from Baltimore to College Park. This move of the 4,000 sq. ft. APC research and education program, including a large aquatic animal facility, has taken approximately 2 years to complete. The move has been a major hindrance to this, and other projects; (2) Change of technical personnel (software programmer); and (3) On a positive note, the ability to secure additional funding from an outside source, in order to add a bacteriology section, has allowed us to broaden the scope the software beyond the original scope of effort. Although the extra development effort resulted in some additional delay, it will ultimately enhance the final product. Development is expected such that CD-ROM disks of the program will be distributed in 2003. IMPACTS: In concise statements (possibly a bulleted list) indicate how the project has or will benefit the aquaculture industry either directly or indirectly and resulting economic values gained (where appropriate). • The new FishGuts software will empower

aquaculturists to make more careful observations relative to the health of their fish stock. This can lead to earlier detection of disease entities and subsequent treatment. Ultimately, reduction in disease-related problems will foster increased production and improved economic gain.

• The new FishGuts software will also foster

greater independence for fish farms. This independence stems from a better ability to take proper samples for fish health and diagnostic purposes. Proper observations, and samples of both tissues and parasites, are key in aiding fish pathologists and veterinarians in making accurate and timely diagnoses.

• Since the FishGuts software is being

distributed throughout the NRAC region with support from this project, and a small “loan collection” of thesoftware will be available through the NRAC office, the information will be available to a larger number of aquaculturists in the NRAC region.

RECOMMENDED FOLLOW-UP ACTIVITIES: State concisely how future studies may be structured. PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED: List under an appendix with the following subheadings: Publications in Print; Manuscripts; and Papers Presented. For the first two subheadings, include journal articles, popular articles, extension materials, videos, technical reports, theses and dissertations, etc. using the format of the Transactions of the American Fisheries Society (example below). Under Papers Presented subheading include the authors, title, conference/workshop, location, and date(s). Manuscripts: We anticipate at least one peer-reviewed publication in an appropriate journal. WWW Outreach through the University of

Maryland Aquatic Pathobiology Center: http://aquaticpath.umd.edu/fg Abstracts (since 1998): Kane, A.S. 2002. FishGuts: An Updated Multimedia

Guide to the Art and Science of Fish Anatomy, Health and Necropsy. Accepted for presentation in the Fish Health Section at the annual meeting of the American Fisheries Society, August 2002, Baltimore, MD. Abstract # 24564429-32

Kane, A.S. 2002. FishGuts: An Updated Multimedia

Guide to the Art and Science of Fish Anatomy, Health and Necropsy. To be presented at the Fourth International Symposium on Aquatic Animal Health, September 2002, New Orleans, LA.

Abstract. FishGuts (updated version 2.0) is a discovery-based, interactive computer program developed to provide training in fish health and necropsy. The program is intended for use by researchers, veterinarians, graduate and veterinary students, fish health professionals, and persons interested in fish anatomy and necropsy. Rainbow trout, bluegill


and channel catfish are used as models to illustrate basic external and internal anatomy. Additional species are included to show inter- and intra-specific variation. Viewers can observe all aspects of a diagnostic examination including case history, gross necropsy, tissue and bacteriology sampling, examination of glass slides and preparation of final reports. Additional sections focus on observing parasites, describing lesions, and microbiological sampling. The new CD-ROM-based FishGuts software contains over 300 MB of digitized sound, narration, photographs, micrographs and QuickTime movies, and serves as an effective, self-paced learning and teaching tool. Viewer-controlled movies are an invaluable tool for identification of live parasites and observing sampling protocols. FishGuts was developed using Adobe Premier and Photoshop, and Macromedia Director software. This project was supported, in part, by the UM Aquatic Pathobiology Center; Maryland Sea Grant; the Northeast Regional Aquaculture Center; The Maryland Agricultural Experiment Station, College of Agriculture and Natural Resources; and numerous contributors and constructive reviewers. Overall Project Objectives. This project involved the expansion and revision of the FishGuts fish anatomy, health and necropsy software, and distributing it throughout the aquaculture community within the NRAC region. Version 1.0 of the existing program was released in 1997. Revisions (i.e., updates) to version 1.0 covered under this proposal included a) the addition of an "aquaculture-friendly" introductory section, b) four new fish species (cultured species within the NRAC region) in the Species Reports section, c) a picture atlas showing grossly visible parasites and lesions commonly observed in cultured finfish, and d) a directory of fish pathologists and diagnostic services available within the NRAC region. The New FishGuts Application. Version 2.0 of the FishGuts software is divided into five navigable sections. These sections, each with separate subsections, include: Section 1: About FishGuts • Updated introduction (new) • How to use the program (an animated narrative) • Program credits and resources including:

1. bibliography (updated) 2. listing of fish health specialists in the

NRAC region (new) Section 2: Anatomy • Differences worth knowing (special sense

organs, poisonous and dangerous fish, intraspecific differences)

• External anatomy (channel catfish model) • Internal anatomy (rainbow trout, bluegill and

channel catfish models) Section 3: Sampling Techniques • Taking a Case History • External examination (non-lethal methods to

examine a fish including anesthesia, general observations, skin scrape, gill biopsy, blood sampling, hematocrit and plasma protein, and gill function animations)

• Internal examination (sacrifice, systematic methods to dissect all organ systems and take samples for preservation and histopathology)

• Bacteriology Sampling (new) Section 4. Case Reports • Weedy Sea Dragon • Channel catfish • Toadfish • Tilapia (new) • Atlantic salmon (new) • Goldfish (new) • Striped bass (new) Sections 5 and 6. What’s My Lesion and What’s My Parasite (new) • Thumbnail views and expanded presentation of different lesions and parasites. Use of the software. Viewers learn how to use the program in the first section, and can take advantage of bibliographic information on fish anatomy, physiology and pathology, as well as look up fish health diagnosticians in the NRAC region. The viewer may then move on to learn about fish anatomy. The use of interactive “rollovers” becomes apparent in the Anatomy section. By rolling the mouse cursor over external and internal anatomical parts of the representative fish, viewers can identify the different


anatomical features. The chapter “Sampling Techniques” reviews the collection of pertinent case history information while taking a relevant account of an animal’s recent background (including water quality and animal- and tank-specific observations). This chapter also contains necropsy subsections with over 50 QuickTime movies that allow the user to view a real necropsy of a rainbow trout. QuickTime movies permit the user full viewing control over movies, including stop action, frame advance, fast forward and rewind. Internal and external necropsy subsections systematically go through sampling procedures for all organ systems. Once the viewer has gone through the first four sections, they can proceed to go through different sample cases. The sample cases are presented with a case history and results of examination procedures. Results include skin scrapes, gill biopsies and gut scrapes for parasites, histopathology, bacteriology, and parasitology (there is a movie of each of the parasites found, with information on parasite taxonomy and the ecological relationship with the host). A final diagnosis is presented for the user to compare their observations with. The Development Process. The development of FishGuts, like any multimedia software application, began with storyboard development. This involved envisioning the “big picture” and putting it into logical divisions or chapters. These chapters were then developed into a series of panels, similar to the tiles of a comic strip. Each tile contained information concerning which media elements (text, sound, narration, animation, picture(s), movies and navigation tools) would be present for each screen display. Development of text and narration, and selection of pictures and movie cuts (subject matter) used in each screen occurred either prior to, or during, storyboarding. The next steps were to develop the new table of contents screen, and modify the program screen layout a user interface. Screen layouts required the capacity to incorporate all of the media elements in a consistent format. In FishGuts each of the program sections were given different color backgrounds to help the user identify with each chapter. To maintain continuity within the program, individual chapter formats remained consistent with regard to layout, fonts and design. The existing interface was maintained as part of the screen layout. The interface gives the user navigation control within the program, i.e., “next screen,” “previous screen,” “go to table of

contents,” “more information” and “quit.” Once chapter and individual screen layouts were adjusted as necessary, media elements were incorporated into the program’s new sections. The software application used for authoring FishGuts was Macromedia Director. Director is just one of many authoring applications available. The Director development platform is somewhat analogous to production of a stage show. Media elements are imported as cast members. Actions of cast members are controlled in a separate score window. Cast members are controlled with a script within the score, which dictates cast member appearance and action on the stage. The stage is synonymous with that which is seen on the computer screen. These concepts, however, are transparent to the viewer when using the final stand-alone software. Storyboard and content development were some of the most important and time-consuming efforts of the FishGuts update. Approximately 8 person-months were spent on storyboard, content development and media acquisition alone. Approximately 12 additional person-months were dedicated to media digitization and manipulation, incorporation into the authoring software (Director), interface and screen design, and interactive scripting and debugging (still ongoing). Using the FishGuts Application. Minimum system requirements for running the new FishGuts software include access to a Macintosh or Intel-based computer (running Microsoft Windows) with a CD-ROM drive, 32 MB RAM, QuickTime (software to run the movies), and a 14" color monitor (640 x 480 pixels or greater). Most Macintosh computers with a CD-ROM drive are likely to be “multimedia smart,” and ready to use. For Windows-based computers, a sound board is also required to take advantage of the audio portions of the program. In general, as with all multimedia CD-ROMs, the faster the computer, the smoother the program will run. Use of a software application, like the updated FishGuts program, does not substitute for gaining actual necropsy experience, nor does it serve as a fish health diagnostic program. However, the software does offer an excellent review of general fish anatomy, methods for making accurate observations, executing a standard fish necropsy protocol,


identifying parasite characteristics, and properly preserving specimens for further analysis. With the increased need for fish culturists, extension agents, veterinarians, and graduate and veterinary students to care for and prepare diagnostic samples from cultured, wild and feral fish for disease analysis, the updated FishGuts software, supported by NRAC, will provide a valuable teaching tool. The new FishGuts software will assist users with making appropriate observations and generating samples for analysis by a trained fish diagnostician or pathologist. Production of this updated FishGuts software will provide a cutting-edge resource to support early disease detection, and will promote the application of appropriate and consistent diagnostic techniques. The Full Report with all the data, graphs and tables is available at the NRAC office upon request.


PROJECT COMPLETION REPORT

97-7 “Developing a sustainable Market for Northeast U.S.A. AquaculturedProducts by Effecting Attitudinal Changes in the Foodservice/Restaurant Sector “

Termination Report Period: March 1, 1997 – May 31, 2001 NRAC Total Funding: $ 46,150 (June 1, 1997 – May 31, 2001) (No-Cost Ext. through June 15, 2000; 2nd through May 31, 2001)

Principal Investigator: Linda O’Dierno, C&J Associates Participating Investigators/ Cooperative Agencies:

Dr. Joe Regenstein Cornell University New York Nancy Kunz C&J Associates New York Chef Demetrios Haralambatos Chef Murray Dychtwald Restaurant Rescue Chef Phil Cragg Institute of Culinary Arts, Atlantic County College Brad Powers Maryland Dept. of Agriculture Maryland Ken Coons New England Fisheries Development Association Massachusetts Chef Joesph Amabile American Culinary Federation New Jersey George Nardi GreatBay Aquafarms New Hampshire Jim Smith United Seacoast Clam Co. New Jersey Stewart Tweed New Jersey Sea Grant New Jersey Leo Dunn Pennsylvania Dept. of Agriculture Pennsylvania

INTRODUCTION Many Americans continue to feel more comfortable eating fish and seafood in restaurants or in other food service settings rather than preparing it at home. It is estimated that between sixty and eighty percent of all fish and seafood consumption takes place outside of the home. In addition, as many other commodity groups have discovered, the restaurant/food service setting has become a natural pathway for the introduction of new foods to the American marketplace and table. Consumers tend to be more adventurous when dining out, each individual can select his/her own entrée which eliminates concerns about different tastes within the group and preparation barriers, i.e. odor problems, don’t know how to cook it, etc., are eliminated. Fish and seafood products are generally understood and better handled within a restaurant venue than in the home kitchen. To a large extent, chefs and kitchen staff have been trained in fish and seafood preparation and are often more familiar with new species than the average home consumer. But, even

the most well-trained chef or food service buyer is sometimes unaware of the many fish and seafood products that are farm-raised and available for purchase.

Aquacultured fish and seafood products are ideal and cost effective for restaurant and other food service applications. To date, their use is not as widespread as it should be in menu planning. Previous studies

courtesy of Jim Avery


(Gall & O’Dierno, 1994) suggested that many food service buyers and suppliers are still unaware of the positive attributes and availability of farm-raised fish and seafood products. Although aquacultured products have inherent advantages that make them ideal for menu planning, food service buyers and chefs are largely unaware of these positive attributes. Among the most important characteristics of the product are consistency of supply, price and quality, all of which are important in developing a satisfied clientele and projecting future profit margins. Food service operators continue to find it difficult to source product especially fish and seafood farmed in the United States. In order for the United States aquaculture industry to compete effectively, it is critical that products be marketed in a manner to develop greater acceptance in the food service sector. This study focuses on ways to position farm raised fish and seafood products in this market. To overcome the difficulties of using farm-raised fish and seafood products that chefs/restaurateurs either experience or perceive, it was necessary to first gain an understanding of their overall view of aquacultured products and then to provide positive, upbeat, accurate information to counteract the morass of misinformation and disinformation that currently exists. The overall objective was to develop a knowledgeable cadre of food service buyers to increase the demand for aquacultured products. A widely held prejudice against aquacultured products is the perceived lack of flavor when compared to wild harvest. This is supported by a number of food writers (see Appendix). There is also a perception, held by both chefs and the general public, that aquaculture operations are not environmentally friendly. This perception was furthered by the publication Murky Waters. Additional concerns about off-flavor or health-related issues exist. Making appropriate fish and seafood menu decisions and maintaining a positive bottom line is dependent on menu item costs. Menu pricing structures ultimately affect the degree of profit. To establish bottom line costs, foodservice managers must accurately calculate center of the plate costs. Drastic price swings experienced by wild harvest fish and seafood products make this process difficult, if not impossible. Inclement weather conditions can prevent commercial boats from fishing, often resulting in a

diminished market supply of fish and seafood. In other instances, boats leave port, enter the fishing grounds only to discover that the expected stocks of fish aren’t there. Boats are forced to return to the dock without the targeted species disappointing prospective buyers. Another factor that interrupting the regular supply of fish and seafood are the state and federal government management regimes that dictate when, where and how much seafood can be harvested. These wild caught fish and seafood supply peaks and valleys further complicate the task of menu planning. Food service managers must often rely on menus that offer “catch of the day” or “market price” options, making it difficult for foodservice operations to: 1) offer fish or seafood products on a regular basis, 2) determine menu pricing and 3) project profit margins. This is unfortunate for both the commercial fishing industry and the aquaculture industry since the majority of fish and seafood consumption (60-80%) takes place outside of the home. The most common scenario employed by the restaurant sector is to replace seafood entrees with chicken alternatives when supply is doubtful and/or the price escalates. Poultry is a natural substitute for the same consumer segment that normally orders fish. This substitution is extremely common in general fare restaurants but more difficult in seafood houses where a greater proportion of the menu features fish and seafood. The practice of commodity substitution results in fish and seafood items appearing sporadically on menus. The customer who experienced a great seafood entrée returns the following week to find it vanished from the menu. The success of fast food chains is based largely on the degree of “sameness” which meets the expectations of customers. A large portion of the public reacts positively to that level of uniformity. On the opposite side, there are adventurous diners that like to sample the catch of the day. The concept of a changing menu also plays to the consumer’s desire for “fresh” fish and seafood. The question remains which of these two segments is contributing the most to bottom line profits. This most likely varies from restaurant to restaurant, and each individual manager will need to make his/her own determination. The correlation most likely occurs in the average entrée price with pricier restaurants willing to offer a


variety of fish and seafood on a regular basis. The background of the diners in upscale establishments may be more cosmopolitan and adventurous in their food choices. Another factor that may be at play is the skill of the chef and kitchen staff in handling a variety of seafood products. Many domestic fish and seafood farmers rely heavily on the ethnic market segment in which product is sold live. Although this is a lucrative and easily accessed market, it is also finite and large-scale production requires expanding into a larger universe of food service markets. METHODOLOGY To address the issues identified above, the research team: 1) assessed the knowledge and use of farm-raised products among chefs/restaurateurs/food service operators in the Northeast, 2) based on those results, developed educational programs/materials including an Aquacultured Products Handling Guide (APHG) to address the knowledge gaps and concerns identified, and 3) conducted a follow-on survey to determine which barriers still existed. An advisory group was created to assist in the selection of aquacultured products and questions to be included in the proposed survey. Background information on the project, a potential list of species for inclusion in the survey instrument and draft questions was sent to 47 individuals including aquaculturists, extension and government personnel, aquaculture associations and chefs in the Northeast. Nineteen individuals agreed to participate on the advisory committee for the project. This group identified the following products in ranked order for inclusion in the survey: trout, oysters, hard clams, tilapia, hybrid striped bass, salmon, catfish, and mussels. Scallops were added to the list at the request of one aquaculture company. The latter was difficult to assess since most restaurants use scallops but many of those scallops are harvested in the wild. Many of the farmed scallops are imported from Asian countries. Trout may also have caused some confusion since wild harvest sea trout is often found on restaurant menus in the region. As a matter of fact, George Washington often dined on grey sea trout at Fraunces Tavern in lower Manhattan. The project’s key priority was the development of user-friendly educational materials that could be incorporated into the Northeast aquaculture community’s plan to develop and expand market

opportunities. The resulting educational booklet, “Aquacultured Products-Recipes and Information for Chefs and Food Service Professionals” featured general information about aquaculture plus detailed information on eight species of fish and seafood farmed in the region. The eight species selected for inclusion were Atlantic salmon, hybrid striped bass, trout, catfish, tilapia, mussels, oysters and clams. The venues for distribution included educational institutions with the intent of familiarizing future chefs with the positive benefits of using farm-raised fish and seafood products. The materials were also distributed to restaurants and other food service establishments. By reading and using the information on the eight farm–raised species, it was hoped that positive attitudinal changes would occur within this sector resulting in the development of a stronger market-demand. These changes could lay the groundwork for additional purchases of aquacultured products and ultimately assist in driving industry expansion. To assist in quantifying the required attitudinal changes, a survey and follow-on survey were conducted over a two-year period. The original survey was conducted prior to the inception of the educational program and distribution of the booklet. The educational effort was based on developing a better market position for aquacultured products and dispelling some of the negative perceptions that were identified in the target audience. The basic message to restaurateurs was that aquacultured products are: 1) consistent in supply, 2) consistent in quality, and 3) consistent in price. These attributes make farm-raised products ideal candidates for the restaurant menu. The inherent stability of the product supply allows for advanced planning and cost/revenue projections. The original premise of the research team was that the most effective way to increase utilization was to focus on the positive attributes of aquacultured products in providing reasonably priced fish and seafood alternatives and improving bottom line profits. The focus on profits was not reflective of the survey results but this might be attributable to how restaurateurs perceive themselves, i.e. more concerned about quality and taste rather than profits. An interesting follow-up would be to conduct some focus groups to delve further into the issue of self-image and how it drives purchases.


Courtesy NJ Dept. of Agriculture

This message was supplemented by information about product forms, actual suggestions as to product use, and pricing examples in the “Aquacultured Products-Recipes and Information for Chefs and Food Service Professionals”. Discussions with restaurant planners, determined that a list of varied suggestions rather than actual recipes would be the best approach since chefs like to develop their own recipes. The proposed suggestion lists for each species were designed to be innovative, and to spark the interest and creativity of the reader. For those who prefer, recipes were formulated and included. Pricing structures and special diet substitutions were provided to demonstrate how these products could be effectively incorporated into almost any menu. To assist chefs in gaining a better overall understanding of aquacultured products, the initial section of the guide was devoted to an explanation of aquaculture and the benefits of farm-raised fish and seafood to the environment and to the consumer. The APHG was developed to be visually attractive with a variety of pictures of locally farmed products. Recognizing the importance that chefs place on their own creativity and menu planning skills, materials in the APHG were designed to be suggestive of different preparation methods to make menus more appealing.

This dish features an oyster stew as an additional menu suggestion. Each section of the Guide provided a list of possible preparations to spur creativity and increase the opportunity to include farm-raised fish and seafood products on the menu.

Trade Show Survey A survey instrument (see Appendix) was developed for use at trade shows throughout the northeast region. The objective of the survey was to identify barriers and prejudices against increased usage of aquacultured product. The survey was conducted at the New York Restaurant Show, the New Jersey Restaurant Show, the Boston Seafood Show, the Mid-Atlantic Food Service Show, the Cook and the Book in Philadelphia, and the Northeast Chapter of the American Culinary Federation Meeting held in Rochester, New York. Mail Survey After distribution of the Aquaculture Product Handling Guide (APHG) and a series of educational programs at the Mid-Atlantic Food Service Show, the New York Restaurant Show, the Cook and the Book Show in Philadelphia, the Northeast Region American Culinary Federation Conference in Rochester, New York, and the International Boston Seafood Show. The follow-on survey was conducted via a direct mail campaign. Each individual completing a survey received a recipe booklet in appreciation. Response to this strategy was much better than had been expected. The survey generated so much interest that individuals actually called to ask questions and request additional information. Based on the response to the mail survey, it would seem that an important way to reach this audience is through educational programs and materials. Many of the restaurant associations and chefs’ groups in the Northeast have regularly scheduled shows and educational events. This is an excellent venue at which to offer educational programs. To further expand the outreach of the program, a copy of the handling guide was provided to Publications International, Inc. This group puts together cookbooklets for broad distribution at supermarkets, bookstores and other outlets throughout the United States. They regularly use fish and seafood recipes. Once a recipe is listed with them, they incorporate it into appropriate publications on an on-going basis. The organization providing the recipe receives credit each time the recipe is used. This is a method to reach large audiences with no additional costs.


TRADE SHOW SURVEY RESULTS The majority (59%) of respondents indicated their entrée prices were in the $15-$20 range. This was common to all sampling venues. Very few respondents were in either the over $25 (6%) or less than $10 (12%) range. The survey did not attempt to segregate out institutional foodservice and restaurant use but some inferences can be made based upon the price categories reported with prices less than $10 being most likely representative of the institutional setting. Among the survey respondents, many local seafood houses along the coast included aquacultured products on their menus and, in some cases, advertised them as such. Based on the responses received from upscale restaurants, $25 and up for entrees, there seems to be a significant prejudice against farm-raised products based on the perceived lack of flavor.

Restaurateurs were asked to indicate which aquacultured products they used and with what frequency. There was clearly some confusion about which products were farm-raised and which were wild harvest. This is evidenced by the number of responses that included question marks in some categories.


Always

Sometimes

Never

Trout

3%

18%

46%

Oysters

18%

40%

72%

Clams

54% (cockles 1)

16%

32%

Tilapia

10%

18%

74%

Hybrid striped bass

3%

18%

82%

Salmon

48%

18%

22%

Scallops

48%

14%

28%

Catfish

0

42%

52%

Mussels**

24%

16%

68%

Sea Bream 2% 0% 92% Arctic Char

2%*

0

92%

Crayfish

0

2%

21%

n=203 *Used arctic char once but then couldn’t get it again. **Many of these respondents were ethnic restaurants primarily Italian, Portuguese and Spanish Percentages are based on the total number of respondents. In some cases, people did not

respond to a specific question.

BEST SELLING AQUACULTURED SPECIES Food service professionals were asked to check off a list of farm-raised fish and seafood products used in their establishments. The volume of usage was denoted by “always”, “sometimes” and “never”. The list of species included was developed by the project advisory committee. In some cases, the researchers felt that the respondent was not aware of whether or not the product was farmed or wild harvest. A large number of respondents included a question mark in the column indicating that they were using a specific product but were unaware of its source. For certain species, like salmon, catfish and trout much of the supply is aquacultured. For other species such as scallops, shrimp, clams, oysters, and mussels, there are likely to be significant amounts of both wild and farmed product commingled. This is especially true in the case of scallops. Thus, the data

above suggests that respondents really do not know the source of their fish supply. A respondent from an Asian restaurant indicated, “ I have no idea what aquacultured product is. Please


explain.” That restaurateur checked the always category for salmon and tilapia. He was also interested in more information on pricing. This was a recall question, and although these lists cannot necessarily be construed as indicating total sales volume, they do provide an indication of which species were most important to each food service operato

Five Top Selling Species Identified by Restaurants Compared to Average Entrée Price >$25 $20-$25 $15-$20 $10-$15 < $10 Salmon Salmon Salmon Salmon Salmon Mussels Oysters Scallops* Tilapia Clams Scallops* Clams Mussels Oysters Mussels Clams Scallops* Clams Catfish Catfish oysters Mussels Catfish Scallops* Tilapia

*these are most likely not aquacultured or if they are ,they are probably imported

Across all price categories, salmon was the number one aquacultured product. This is most likely a result of consumer acceptance coupled with current price structure which makes it acceptable to the various margins required by different types of operations and the increased availability of pin-bone-out fillets. It is interesting to note that mussels, probably because of their cosmopolitan appeal, were used in the many upscale restaurants. Although scallops were reported in all price categories except the less than $10 category, these were most likely wild harvest or cultured in Asia.

When asked to identify the perceived advantages of aquacultured products, consistent quality (46%)

and supply (42%) were clearly the most important attributes reported. This may be a factor of the prompts in the survey combined with the respondent’s self perception, i.e. quality is my most important concern. In developing overall marketing strategies, this may be an important promotional point. Consistent price (4%) and lower price (8%), attributes that were deemed important by the researchers, were rated much lower by the user community. Safety concerns (16%) still played an important role in the user’s overall product perception although that response was lower than in previous studies. Better shelf life (14%) and better portion control (12%)

0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50%

Percent of Respondents

Consistent Supply

Consistent Quality

Consistent Price

Consumer Perception of Quality

Safety Concerns

Better Shelf Life

Lower Cost

Better Portion Control

Attr

ibut

es

Perceived Advantages of Aquacultured Products


were also important. It should be remembered that respondents may reflect what they want the world to think and/or what they think the survey team wants to hear. By developing an understanding of how the buyer perceives the product, more effective strategies can be developed to position product in the marketplace. The importance of consistent supply is reinforced by the buyer’s inability to purchase product on a regular basis. This can be attributed to lack of a consistent source of volume production. Many aquaculture operations in the region are small and harvest on an infrequent basis. This negates the advantage of consistent supply. A small farmer harvests and sells his/her product and then begins a new growth cycle. Catfish has gained prominence not only because of the product attributes (white, mild tasting, boneless fillet) but also the availability of a consistent supply of product. Consistent supply was identified as a primary advantage (42%). However when respondents were asked to provide information about disadvantages, the major constraints were the lack of regular supply and the small number of wholesalers carrying aquacultured products. This is in direct contradiction to the perceived advantages. This is an important

concern that must be addressed by aquaculturists as they develop markets. This concept was reinforced in the follow-on survey. Several suppliers completed the survey and indicated that the only farm-raised product that they handled was salmon and they did not promote it as an aquacultured product. One user indicated that he was “not aware of any advantages.” One individual from a shoreside restaurant concluded, “In my experience most farm-raised products do not have the flavor of “wild” or products captured in their natural habitat.”

0% 5% 10% 15% 20% 25% 30%


Unable to Get a Regular Supply

Wholesaler Doesn't HandleAquaucltured Products

Less Flavorful

Limited Variety

Attri

bute

sPerceived Disadvantages of Aquacultured Products


Incentives When asked to identify incentives for the promotion of aquacultured products, there was clearly an interest in the development of table tents and menu clip-ons. The Alaska Seafood Marketing Institute regularly uses rebates and incentives targeted at suppliers and supermarkets. Since prompts were used in this question, there was also considerable interest in rebates.

Restaurateurs also perceived live tanks as a positive incentive that could increase sales. This perception may be a result of lobster tanks, which help to connote product freshness. When using live tanks with finfish, there is an increased need for skilled food handlers and this may not part of consumers who may be squeamish about selecting a live fish.

Educational materials are aimed at the foodservice and restaurant staff to help them understand what farm-raised fish and seafood are and why they should be using them. Rebates are desirable because they put money back into the pocket of the restaurant or foodservice operation. Menu clip-ons, table tents and live fish tanks are aimed at generating customer interest and ultimately sales. Therefore, to compare these “incentives” to each other may not be doing justice to continuing the educational aspect of developing a larger market share for farm-raised products.

USE OF THE TERM FARM-RAISED AS A MARKETING TOOL Very few restaurants used the term farm-raised as a marketing tool. This is in direct contrast to the 1980’s when there were significant concerns about pollution and product safety. At that time, cultured products were perceived to have a decided advantage over wild-harvest. Given the barrage of concerns about food safety, consumers have become numb to those issues.

0% 2% 4% 6% 8% 10% 12% 14% 16%


Special Promotions

Menu Clip-Ons and Table Tents

Sales Incentives

Tours of Supplier Facilities

Rebates

Educationsla Materials

Live fish Tanks

Ince

ntiv

e

Incentives to Include More Aquaucltured Product on the Menu


Use Term Farm-Raised

Use of Farm-Raised as aMarketing Tool

6%

90%

4%

Do Not Use Term Farm-Raised

NoResponse

Results of the Follow-on Survey After Delivery of Educational Program After completion of the initial survey, an educational campaign was mounted. The campaign consisted of participation at trade shows and professional meetings in the northeast where food service professionals learned more about aquacultured products and were provided with a copy of the handling guide.

The objective was to create attitudinal change among users and develop a greater acceptance of aquacultured products in the foodservice sector. Positive attributes of aquacultured products were stressed as well as suggestions for including those products in the overall menu planning strategy. The majority (50%) of restaurants responding to the follow-on survey were in the $15-$20 entrée price category. There were also responses from fast food chains where the respondent wanted to increase his/her knowledge about the product. Although these outlets do not use aquacultured products, these individuals may move on to other management positions where they can make or influence purchasing

decisions.

Percentage of Restaurants/Foodservice OperationsResponding in Each Entree

over $25

$20-$25

$15-$20

$10-$15

Less than $10


Frequency of Use of Farm-Raised Products by Percentage of Respondents

Always Sometimes Never Trout 14% 52% 25% Oysters 28% 34% 28% Clams 33% 39% 20% Tilapia 14% 43% 27% Hybrid Striped Bass 25% 25% 28% Salmon 61% 29% 12% Scallops 41% 30% 23% Catfish 18% 35% 38% Mussels 32% 36% 15% Sea Bream 1% 12% 33% Arctic Char 3% 17% 56% Crayfish 5% 21% 47% n=191

Frequent use of hybrid striped bass has moved up appreciably from 3% in the initial survey to 25% in the follow-on survey. Frequent oyster use increased from 18% to 28%.

This may be a factor of the time during which the two surveys were conducted and seasonality of the product. Catfish went from 0 to 18% in the frequent use category. This may be attributed to extensive and well placed industry promotion.

.Five Top Species Identified by Restaurants Compared to Average Entrée Price

>$25 $20-$25 $15-$20 $10-$15 < $10 Salmon Salmon Salmon Salmon Salmon Scallops Oysters Mussels Tilapia Catfish Mussels Clams Clams Clams Mussels Clams Mussels Catfish Oysters Oysters Trout Trout Tilapia Catfish Tilapia

As entrée price decreased, there was a marked decrease in the use of aquacultured fish and seafood products. Sixty percent of the respondents indicated an infrequent use of these products. Catfish has moved up significantly in the mid-priced restaurants. Many foodservice professionals are still not aware of the source of their fish and seafood offerings. One individual in the $10-$15 entrée price category indicated, “We do not use farm-raised fish. These are the seafoods that we always use--salmon and mussels.” A second individual indicated that he was unaware of the method of the harvest. Another respondent indicated that the only product on the list that he was sure was aquacultured was tilapia.

Several large corporate cafeterias responded that they sometimes use catfish and tilapia. One respondent indicated that since his is a chain restaurant, purchase decisions and menu planning are handled centrally.


0% 10% 20% 30% 40% 50% 60% 70% 80%


Consistent Supply

Consistent Quality

Consistent Price

Consumer Perception of Quality

Safety Concerns

Better Shelf Life

Lower Cost

Better Portion Control

Attr

ibut

es

Perceived Advantages of Aquacultured Products

A significant increase in the promotion of product as farm-raised occurred during the interval between the initial survey and the follow-on survey. In 1998, only 6% of the respondents indicated that they used the term “farm-raised” while in 2001 that number increased to 30%.

Several respondents indicated that they use the term “farm-raised” on the menu and that it is preferred to “aquacultured”. In the follow-on survey, consistent quality (74%) was again listed as the most important attribute. This was an increase over the initial survey in which only

Use Farm-Raised as aMarketing Tool

5%

65%

30%

No Response Do Not Promote as Farm-Raised

Promote as Farm-Raised


(46%) of the respondents listed this attributed. Consistent supply (69%) was the second most important attribute. This is similar to the initial survey (42%). Consumer perception of quality was not an important attribute in either survey. Concerns about price increased from four percent (4%) to forty-six percent (46%) as can be expected in a tightening economy. Two respondents indicated that the price of aquacultured products fluctuate as severely as wild product. Both of these users were actively purchasing aquacultured species (salmon, trout and tilapia) and were promoting the products as being farmed raised. The price fluctuations in these species categories may be attributable to the number of suppliers.

One individual indicated that consistent size was an advantage. This may be a slight difference from portion control since it may be whole fish that are being purchased. Only one respondent indicated that an advantage was to “ensure the longevity of the species.” One individual indicated “really like the flavor of cultured mussels. Also I feel the texture is superior to regular or Chinese mussels.” According to one respondent, “Chilean salmon has a better taste profile.”

Perceived Advantages by Entrée Price Category Under $10 $10-$15 $15-$20 $20-$25 Over $25

Consistent Quality (70%)





Consistent Price (52%)

Consistent Supply ( 80%)

Consistent Supply (69%)








Consumer Perception of Quality (26%)

Safety (33%) Safety (50%) Consumer Perception of Quality (43%)

Portion Control (12%)

Safety (20%)


Better Shelf Life (31%) Safety (29%) Safety (9%)

Shelf Life (18%) Lower Cost (25%) Better Portion

Control (31%) Lower Cost (14%) Shelf Life (5%)

Portion Control ( 23%)

Better Shelf Life (6%)




Lower Cost (7%) Consumer Perception of Quality (0%)

Lower Cost (19%) Better Shelf Life (5%) Lower cost (0%)


0% 2% 4% 6% 8% 10% 12% 14% 16% 18%


High Price

Unable to Get a Regular Supply

Wholesaler Doesn't HadnleAquaucltured Product

Less Flavorful

Limited Variety

Attr

ibut

esPerceived Disadvantages

Very few respondents listed disadvantages. This can be attributable to their lack of knowledge and familiarity with the products. It might also be a factor of reticence to be negative about the products. One individual did raise a concern about water quality. According to one chef, “the largest drawback is the lack of high quality flavor, eg., farmed trout vs, stream trout.” Another major disadvantage was the lack of availability. Incentives One individual in the over $25 entrée price category indicated, “make the species more like wild—stop feeding pellets, give them bigger tanks, feed them things they would more likely eat in nature. Anything that would make them more like wild.” There was a significant amount of interest in educational materials and this interest in reinforced by the response to the direct mail survey in which there was a 23% return rate. Three individuals called as a result of the survey to ask more questions. As a result of that interest, a brief power point presentation was developed.

As was anticipated, rebates also scored high as an incentive. Special promotions were rated high. This might be an opportunity for a small farmer to work with a local restaurant or for a large supplier to do a concentrated promotion, i.e. national catfish month. Such promotions might appeal to “sometimes” users.


IMPACTS The overall project impact was, and continues to be, the development of a cadre of institutional/restaurant buyers and chefs that possess a greater understanding of the attributes of aquacultured products and the important contribution of these products to successful and profitable menu development. It is hoped that these efforts will result in increased market opportunities and sales for Northeast U.S.A. aquaculture producers. The production of the publication provides a strategy to continue to reach the audience beyond the scope of the initial project. The survey did not attempt to segregate institutional food service versus restaurant use but some inferences can be made based upon the price categories reported with prices less than $10 being most likely representative of the institutional setting. Use in some institutional settings such as cafeterias provides a very viable way to provide educational materials to the consumer to encourage future retail purchase. Efforts should be made to introduce aquacultured products to other food service outlets

especially school lunch programs that can help to develop life-longer fish and seafood eaters. One of the important issues uncovered by the survey was that food service suppliers were not actively promoting aquacultured products. The suppliers who responded to the survey indicated that the only aquacultured product that they were handling was salmon. Only one of the food service suppliers actively promoted the product as being farm-raised. Educating the food service supplier can be a significant multiplier since that knowledge may flow down to the individual restaurant and ultimately to the retail consumer. An important marketing channel for aquacultured products is an increase in the array of products being offered by foodservice suppliers. A large number of respondents indicated that they were unable to get a regular supply of product. This is most likely a factor of the low level of production in the region and the presence of many small farmers. Many foreign nations can easily access our domestic markets because of the large volume of product that is being

0% 5% 10% 15% 20% 25% 30% 35% 40%


Special Promotions

Menu Clip-Ons and Table Tents

Sales Incentives

Tour of Supplier Facilities

Rebates

Educational Materials

Live Fish Tanks

Ince

ntiv

es

Incentives to Include More Aquacultured Product on the Menu


produced, the low cost and the government-supported effort to generate hard currency. SUMMARY ACTIONS • The major disadvantage identified by

foodservice professionals was the lack of a regular supply of product. This is most likely a function of the low level of production in the region and the industry profile consisting largely of small farmers.

• Low production levels preclude sales through

major foodservice distributors. • The most effective marketing channel for the

small grower is most likely individual restaurants and/or small distributors/wholesalers.

• If sufficient supplies can be developed, it is

important to work closely with the foodservice supply network to promote the advantages of aquacultured product and counteract the misinformation and disinformation that is sometimes put forth about American aquaculture.

• Based on the interest sparked by the project, an

effective marketing channel would be greater participation in foodservice related meetings, events and small trade shows where growers can interface with buyers on a one to one basis.

• Since several large corporate foodservice

providers indicated that they use catfish and tilapia, this might be a good opportunity to distribute consumer cookbooks to help generate retail sales. Some corporate cafeterias regularly do employee promotions.

• Supply was identified as a major impediment to

using aquacultured products. To address this issue, it might be beneficial to form marketing groups so that supply can be increased.

• One of the disadvantages cited by several of the

upscale restaurants is the lack of flavor compared to wild stocks. It would be beneficial to do some blind taste tests with this audience.

• A possible mechanism to counteract the sporadic

supply, might be special promotions such as

“oysters R here”. Promotions were regarded as an important incentive by the respondents.

LITERATURE CONSULTED Consumer Expenditure Survey, 1990-1992. 1993.

US Department of Labor Statistics, Washington, DC 20202

Fisheries of the United States, 1999. National

Marine Fisheries Service, Washington, D.C. Gall, K and L. O’Dierno, “Aquaculture Marketing

Survey”, Northeast Regional Aquaculture Center, No. Dartmouth, MA, 1994.

Goldberg, R. and T. Triplett, 1997. “Murky

Waters—Environmental Effects of Aquauclture in the United States” Environmental Defense Fund, New York.

Fisheries of the United States, 1999. National

Marine Fisheries Service, Washington, D.C. The Full Report with all the data, graphs and

tables is available at the NRAC office upon request.


APPENDIX

Examples of popular food press focusing on perceived negatives of farm raised fish and seafood


PROJECT COMPLETION REPORT 98-4A “Determination of Optimal Conditions for Swimbladder Inflation 98-4CE in Striped Bass Larvae Reared in Intensive Systems” Termination Report Period: March 1, 1999 through August 15, 2000 NRAC Total Funding: $84,642 (March 1, 1998 – Febuary 28, 2000) (No-Cost Ext’n through August 15, 2000) Principal Investigator: William F. Van Heukelem, University of Maryland, Center for Environmental

Science, Horn Point Laboratory, P.O. Box 775, Cambridge, MD 21613 Participating Investigator/ Cooperative Agencies:

Reginal M. Harrell University of Maryland Maryland Steven G. Hughes University of Maryland Maryland Scott Lindell AquaFuture, Inc. (now Fins Technology) Massachusetts Bruce R. Friedmann AquaFuture, Inc. (now Fins Technology) Massachusetts

REASON FOR TERMINATION Objectives were completed and funds were spent for project.

PROJECT OBJECTIVES: The overall objective of this project was to determine the optimal conditions for increasing numbers of striped bass larvae with inflated swimbladders in intensive culture systems and to disseminate the information obtained to producers in the industry. Specific objectives for the two year study at the three sites were as follows: Year 1, In 70 liter tanks at Horn Point Laboratory (HPL), Maryland. 1. Determine optimum light intensity for

swimbladder inflation. 2. Determine optimum water turbidity and light

level for swimbladder inflation. 3. Determine effect of spray bars on swimbladder

inflation. 4. Present results at a national Aquaculture meeting. Year 1, In beakers at University of Maryland Eastern Shore (UMES). 1. Determine effects of three surfactants on

swimbladder inflation.

Year 2, HPL. Using a combination of optimal conditions of light intensity, turbidity and spray bars found during the first year of the study at Horn Point: 1. Determine if the best surfactant found in year 1

studies in beakers at UMES is of added benefit in 70 liter tanks.

2. Determine if the best combination of factors found in the preceding experiments are equally effective when used in production scale ( 900 and 2,000 liter) tanks.

3. Verify that optimal conditions determined in prior experiments work with larvae from five additional broods of larvae.

4. Present results at a national aquaculture meeting 5. Prepare a fact sheet on optimal conditions for

swimbladder inflation 6. Prepare results for publication in a scientific

journal. Year 2, UMES. 1. Determine effects of antibiotics on swimbladder

inflation. 2. Determine effects of water hardness on

swimbladder inflation.


Year 2, AquaFuture, Massachusetts: 1. Determine effectiveness of techniques developed

at HPL and UMES under “real world” production conditions at a different facility.

Year 3, HPL (Drs William Van Heukelem and Reginal Harrell) 1. Determine maximum stocking density that will

still produce high rates of swimbladder inflation. 2. Determine optimum water hardness for

swimbladder inflation based on additional research to be performed at UMES.

3. Determine if new surfactants pre-tested at UMES in 2 liter beakers can increase swimbladder inflation in our larger tanks.

Year 3,UMES (Dr. Steven Hughes) 1. Expand previous experiments to determine

optimum water hardness for swimbladder inflation and survival including the interaction of hardness with chlorides or magnesium.

2. Test new surfactants and concentrations for effects on swimbladder inflation and survival. Medical surfactants which are used with respiratory patients and premature infants will be tested.

Year 3, Fins Technology (Scott Lindell) 1. Test best conditions for swimbladder inflation as

found at HPL and UMES under “real world conditions” at a commercial facility. They will use three broods of larvae and compare results with those obtained using their normal rearing conditions.

Year 3 Outreach We will present results at Aquaculture ‘01 as well as in a publication and will prepare a new fact sheet on optimal conditions for swimbladder inflation Results obtained from this project will be presented at a national aquaculture meeting at the end of each year of study. Results will also be published in a scientific journal at the end of the study. In addition, a fact sheet will be developed and distributed and made available for publication on the NRAC Home Page on the World Wide Web. ANTICIPATED BENEFITS: This project will increase our understanding of conditions that must be provided to produce batches

of striped bass larvae with a high percentage of inflated swimbladders. Results should be applicable to hybrid striped bass also as well as other species cultured in intensive systems. Using the techniques learned from our studies, researchers and producers will be able to produce more larvae from each spawning that are viable, grow well and are free from lordosis that is caused by uninflated swimbladders. PRINCIPAL ACCOMPLISHMENTS: Year 1 (1998) Three experiments were performed in April 1998 in five independent recirculating systems each containing ten 70 liter tanks. Five replicate tanks were used to test the effects of each variable in the first two experiments. In the first experiment the effects of light intensities of 0, 10, 25, 50, 100, 250, 500, 1000 and 2000 lux were studied. The percentage of larvae with inflated swimbladders was highest in the two lowest light intensities but there was a block effect among the different systems that clouded the results to some extent. In the second experiment effects of Aquashade concentrations 0, 2, 4, 8 and 16 ppm were tested at two light intensities ( 1 and 10 lux). There was no difference in the percentage of larvae with inflated swimbladders at any of the tested concentrations of Aquashade or at either light level. The percentage of larvae with inflated swimbladders averaged 98% in all treatments. Although we planned to study the effects of spray bars in the third experiment, upon reviewing our preliminary results from 1997 (funded by HPL) it was clear that spray bars did not increase the percentage of larvae with inflated swimbladders. Therefore, instead of repeating that experiment we decided to examine the effects of direct verses indirect light at a low level of intensity. In the third experiment the effects of direct verses indirect light were studied. Light intensity at the surface of each tank was maintained at 15 - 20 lux. A total of 40 tanks were used and half of the tanks received direct lighting whereas the other half received indirect light bounced off the ceiling. There was no difference in swimbladder inflation between tanks receiving direct vs indirect light.


Three surfactants (Span 20, Witconol 14, and Glucopon) were tested as surface sprays in two-liter beakers using stock solutions of 10, 100 and 500 ppm. Only Witconol 14 showed promise as an aid to increasing swimbladder inflation in striped bass larvae and only at the lowest concentration tested. Survival in the 10 ppm Witconol treatment was slightly lower than in the control treatment and swimbaldder inflation showed a significant (P<0.05) increase. Results were presented at Aquaculture Americas ‘99 in Tampa, FL, January 1999. Year 2 (1999) Three experiments were performed at HPL in April 1999 in five independent recirculating systems each containing ten 70 liter tanks. Larvae were stocked in tanks at a density of 30 /L (2,100 per tank) at three days post hatch and were reared at 18oC and 3 ppt salinity. The percentage of larvae with inflated swimbladders was determined on day 8 post hatch. In experiment one, the effects of ten light intensities from 0 to 2000 lux were studied in five replicate tanks per light level. Light intensities were held constant during this experiment. The percentage of larvae with inflated swimbladders was highest in constant dark (0 lux). Similar results were obtained at AquaFuture, Inc. (Turners Falls, Massachusetts) in covered vs uncovered 125 gal tanks. In experiment two, effects of two surfactants and a 12:12 light cycle were tested. The use of surfactants (Witconol 14 and Glucopon) did not increase swimbladder inflation and the 12:12 light cycle was detrimental to swimbladder inflation when compared to constant dark. In experiment three, swimbladder inflation was studied in 5 different broods of larvae using the best conditions found during the previous experiments. All tanks remained constantly dark (0 lux) and a total of 40 tanks were used. Half of the tanks were sprayed with a surfactant solution (Glucopon at 5 ppm) twice daily. The other 20 tanks served as controls (no surfactant). There was no difference in swimbladder inflation between the surfactant treatment and control. One brood of larvae had significantly lower swimbladder inflation than the others.

Experiments at the University of Maryland Eastern Shore in 2 L beakers showed that the surfactant Witconol 14 enhances swimbladder inflation. The effects of three antibiotics on swimbladder inflation were also studied and none were beneficial. Increasing calcium content of rearing water to 250 ppm resulted in higher swimbladder inflation rates. Swimbladder inflation in production scale tanks (2000 L) at HPL was poor in comparison to that obtained in the 70 L experimental tanks. We believe that the poor inflation in these tanks was due to high densities of eggs and larvae (resulting in heavy oil films) and low water hardness. Results were presented at Aquaculture Americas 2000, in New Orleans, LA, February, 2000. A fact sheet (NRAC Publication No. 00-006) was produced in May, 2000. Year 3 (2000) Several experiments were performed at HPL in April 2000 in five independent recirculating systems each containing ten 70 liter tanks. Separate experiments also were performed in 1000 L and 2000 L tanks to determine if results obtained in small tanks could be obtained in larger tanks. Larvae were stocked in tanks at a density of 30 /L and 60/L at three days post hatch and were reared at 18 - 20oC and 0 or 3 ppt salinity. The percentage of larvae with inflated swimbladders was determined on day 8 post hatch. Effects of water hardness were studied by rearing larvae in fresh well water. Calcium hardness was increased by adding calcium chloride to obtain hardness of 20, 70, 120 and 170 mg/L. Swimbladder inflation was somewhat better at higher hardness levels (95 % at 170 mg/L versus 91 % at 20 mg/L). Larvae did equally well at densities of 30 and 60 /L in 70 L black cones but swimbladder inflation was better at the lower density in 1000 L tanks. Results in 1000 and 2000 liter tanks were mixed. By covering tanks with black plastic we were able to obtain better swimbladder inflation than we had in previous years, but we could not consistently obtain the high inflation rates ( > 90%) in our large tanks that we obtained in 70 L black cones. Results will be presented at Aquaculture ‘01 in Orlando, FL in January, 2001.


IMPACTS: Using the techniques learned from our studies, researchers and producers will be able to produce more larvae from each spawning that are viable, grow well, and are free from lordosis that is caused by uninflated swimbladders. RECOMMENDED FOLLOW-UP ACTIVITIES: Because oil on the surface of the water (from decomposing eggs and larvae) prevents some larvae from penetrating the surface of the water to obtain air for initial swimbladder inflation, the search for effective, non-toxic surfactants should continue as these hold the most promise for increasing the percentage of larvae with inflated swimbladders in high density culture. Turbidity, produced by adding clay to the water, has proved to be an aid in swimbladder inflation in walleye larvae but has not been investigated in striped bass larvae, and this should be done. More research should also be done on the relationship of water hardness, both calcium and magnesium, to swimbladder inflation. Our research has focused on striped bass but we think that the techniques should also apply to hybrid striped bass larvae as well as unrelated species. Experiments should also be performed to verify our belief that the techniques we used will work with other species. PUBLICATIONS:

Van Heukelem, W.F., J.M. Jacobs, R.M. Harrell,

S.G. Hughes, S. Lindell and B. Friedmann. Optimal conditions for swimbladder inflation in striped bass larvae reared in intensive systems. Northeastern Regional Aquaculture Center Publication No. 00-006 (May, 2000).

PAPERS PRESENTED:

William F. Van Heukelem, John M. Jacobs, Reginal

M. Harrell, and Steven G. Hughes. 1999. Effects of Light Intensity, Aquashade, and Surfactants on Swimbladder Inflation in Larval Striped Bass. Aquaculture America ‘99, January 27 - 30, 1999, Tampa, Florida

William F. Van Heukelem, John M. Jacobs, Pilantana

Anderson, Megan O’Connor, Steven G. Hughes, Scott Lindell, Bruce R. Friedmann, and Reginal M. Harrell. Effects of Light Intensity, Photoperiod, Water Hardness, and Surfactants on Swimbladder Inflation in Larval Striped Bass.

Aquaculture Americas 2000, February 2 - 5, 2000, New Orleans, Louisiana.

Paper to be presented at Aquaculture ‘01 January

21-25, 2001, Orlando, Florida: William F. Van Heukelem, John M. Jacobs, Pilantana Anderson, Megan O’Connor, Steven G. Hughes, Scott Lindell, and Reginal M. Harrell. Swimbladder Inflation in Larval Striped Bass: Effects of Water Hardness, Larval Density and Tank Size.

The Full Report with all the data, graphs and tables is available at the NRAC office upon request.

Northeastern Regional Aquaculture Center 40 Annual Progress Report 9/1/99 - 8/31/00

PROJECT COMPLETION REPORT 98-6 “Development and Application of Multiplex PCR for Screening of Shellfish Pathogens” Termination Report Period: July 21, 1999 – May 31, 2001 NRAC Total Funding: $142,103 (September 1, 1998 – May 31, 2001)

Principal Investigator: Dr. Richard A. French, University of Connecticut Participating Investigators Mazhar Khan University of Connecticut Connecticut Roxanna Smolowitz Marine Biological Laboratory Massachusetts John Volk Connecticut Department of Agriculture Connecticut John Karolus Connecticut Department of Agriculture Connecticut Inke Sunila Connecticut Department of Agriculture Connecticut Lynn Hinckley University of Connecticut-DTS Connecticut Marianne Kalbac University of Connecticut Connecticut Spencer Russell University of Connecticut Connecticut Hillard Bloom Tallmadge Brothers, Inc. Connecticut Robert Smith Rhode Island Clam Co. Inc. Rhode Island Karen Eno Aeros Cultured Oyster Co., Inc. New York Steve Fleetwood Bivavle Packing Company, Inc. New Jersey REASON FOR TERMINATION: Objectives are completed and the funding period has ended PROJECT OBJECTIVES: l. To develop and optimize a multi-species polymerase chain reaction (PCR) (multiplex PCR) for the detection of the eastern oyster (Crassostrea virginica) pathogens Haplosporidium nelsoni (MSX), Haplosporidium costale (SSO) and Perkinsus marinus (Dermo). 2. To determine if the multiplex PCR can be used as a specific and quantitative measure of infection by the oyster pathogens H. nelsoni (MSX), H. costale (SSO) and P. marinus (Dermo) . 3. To apply the technology (multiplex PCR) to field samples and or animals at point sources of infection and to evaluate and compare findings with conventional testing procedures. 4. To determine if the specificity and sensitivity of the multiplex PCR method can provide accurate and meaningful data for use as a diagnostic aid in the management of H. nelsoni (MSX), H. costale (SSO) and P. marinus (Derrno) in the eastern oyster.

ANTICIPATED BENEFITS: Product: A single, rapid multiplex PCR test for H. nelsoni, H. costale and P. marinus in the eastern oyster (Crassostrea virginica). User/Beneficiary: The product will have potential use in commercial and private industry, research, diagnostics, and in certifying and regulatory laboratories. The products are primarily expected to serve the aquaculture industry in the routine screening of oyster seed-stock. In this context, certifying will be possible and the management of disease in the cultivation of oysters can be engineered. Economic benefit: The introduction of multiplex PCR for testing of disease pathogens is expected to increase product availability & quality by increasing disease pathogen monitoring and thus decreasing endemic disease and disease impact. PROGRESS AND PRINCIPAL ACCOMPLISHMENTS: OBJECTIVE 1: - Completed. Results are submitted for publication (see attached manuscript). OBJECTIVE 2: - Completes. Results are submitted for publication (see attached manuscript).


- Dr. Soledad Penna conducted the research and defended her masters thesis in May 2000. OBJECTIVE 3: - To be completed, September 2001 In 1999, a multiplex polymerase chain reaction (MPCR) was developed in the Department of Pathobiology, University of Connecticut for the simultaneous detection of one member of the Phylum Apicomplexa, Perkinsus marinus (Dermo), and two members of the Phylum Ascetospora, Haplosporidium nelsoni (MSX) and Haplosporidium costale (SSO). After initial testing and review of the initial multiplex PCR of Penna et al., (manuscript attached) improvements were necessary to address quality control and sample variability. To further improve the MPCR and to test its validity, researchers from the Department of Pathobiology have collaborated with Dr. Inke Sunila of the Connecticut Department of Agriculture, Milford Bureau of Aquaculture and Laboratory (Milford, CT.), Dr. R. Smolowitz of the Marine Biological Laboratory (Woodshole, MA.) and numerous other institutions and commercial aquaculture enterprises along the east coast and the Gulf of Mexico. The objective of the project is to validate the new and improved MPCR by comparing its results to conventional diagnostic procedures used today. To complete this objective, a one year research project has been designed to 1) test oysters collected from 10 different Atlantic coast States and 2) test and monitor two commercial oyster production sites within Long Island Sound bimonthly for one full year. In addition we can utilize the samples to further explore molecular diagnostic technology and to develop other specific and sensitive diagnostic tools. MPCR redevelopment and Field Trials

1. To introduce a 'quality control' gene into the previously developed multiplex polymerase chain reaction (MPCR) for the detection of an 805-bp segment of 28s rRNA gene of Crassostrea virginica, as well as the oyster pathogens Haplosporidium n elson i (MSX) , Haplosporidium costale ( S SO) , and Perkinsus marin us (Dermo) in the eastern oyster. 2. To apply the MPCR to field samples and to evaluate and compare findings with conventional testing procedures.

OBJECTIVE 2: - Completed. Results are submitted for publication (see attached manuscript). The goal of this experiment is to produce a diagnostic test that accurately indicates the presence or absence of each pathogen in any given oyster sample. False positive reactions due to either cross contamination,

non-specific crossreactivity and the action of PCR inhibitors can result in partial or complete failure of the PCR to occur (Brightwell, et al 1998). With the increased in reliance on rapid PCR based diagnostic tests for pathogenic micro-organisms it is becoming imperative to incorporate internal standards to the PCR Assay to assure accurate diagnosis and to avoid false positives and negatives (Brightwell, et al 1998). A PCR internal standard is a DNA fragment co-amplified with the target PCR products, which is of different size than the target products (Sachadyn et al 1998). To address this need, an 805-bp specific sequence product from 28s rRNA gene of C. virginica has been successfully incorporated into the MPCR to act as an internal, 'quality control' product in our reaction. The positive visualization of an oyster-specific amplicon of greater molecular weight than target amplicons within each sample tested confirms the quality of the DNA isolated for application of the MPCR, and represents DNA amplification under optimized conditions for the primers applied. RESULTS TO DATE 1. Successfully incorporation of a quality control gene into the MPCR and tested for its Specificity and Sensitivity. 2. Twenty-five samples of thirty oysters each have been processed and tested. Each oyster is subjected to the newly developed MPCR and the conventional diagnostic methods; RayMackin Fluid Thioglycollate Medium assay and Histology. 3. Sampling sites to date: • Massachusetts • Rhode Island • Connecticut • New York State • New Jersey • Maryland • South Carolina • Louisiana • Washington State • Maine OBJECTIVE 4: - To be completed, October 2001 Evaluation of the results and testing procedure will be conducted with industry and state agencies collaborating in the study. Studies have been perforrned at of the Connecticut Department of Agriculture, Milford Bureau of Aquaculture and


Laboratory (Milford, CT.) by Dr. Inke Sunila, and the Marine Biological Laboratory (Woodshole, MA.) by Dr. R. Smolowitz. The Bureau of Aquaculture reports good results and reproducibility with the MPCR methods and Dr. Sunila is working closely with our group to finalize field trials that have produced excellent results (to be published, see Manuscripts in Preparation). Dr. Smolowitz laboratory (Woodshole, MBL) had difficulties with the assay which are summarized below. We are moving forward in modifying the test to make it more user-friendly and have been awarded a grant from National Sea Grant Of fice (Development and Application of a Chemiluminescent Quantitative Multiplex PCR for Summary of Dr. R. Smolowitz (subcontract): FIELD TRIALS USING MULTIPLEX PCR AS A METHOD OF BIVALVE DISEASE DIAGNOSIS AT THE MBL Roxanna Smolowitz, DVM Joe Cavanaugh, Technician The objective of this study was to explore and test the use of the multiplex PCR diagnostic method, developed by Dr. Richard French and his associates at U. Conn., in another bivalve diagnostic laboratory. Funding, methods and appropriate reagents were provided in Feb. 2001 from NRAC/U. Conn. and testing occurred over a 5 month period from that date. Tissues used for testing in this method originated from oysters collected from two locations on Martha's Vineyard, MA (SSO and Dermo positive stock) and one location in Dennis, MA (Dermo and MSX positive stock). In addition to examination using multiplex PCR, animals were examined histologically and/ or with thioglycollate culture for these three diseases. Results of the PCR testing were compared to the more traditional testing methods. Conclusions and Recommendations for Future Testing of Multiplex PCR: Because of the problems in the establishment and use of the Multiplex PCR method in our laboratory, we were not able to do any real comparison between traditional and multiplex PCR diagnostic tests for sensitivity and reproducibility. However, based on our short and limited experience with this test, we do have recommendations for use in its further development, especially if it is to be used as a standard testing method in various diagnostic laboratories. 1. Quantify the DNA concentrations of several individual oysters taken from different areas to determine the average variance between individuals and areas. (Pl Comments: This procedure is routinely

performed in the laboratory of the Pl and is requiredfor standardization of laboratory results) 2. Sample oysters at the appropriate time of year when the various pathogens are known to be present to increase the number of expected positive individuals tested. (Pl Comments: The testing procedure is being tested over the course of two full years and includes more than 12 sites {see objective 3, above2. The goal is to cover periods of low and high disease prevalence and regions of variable disease epizootiology) 3. Do further work to establish the best location from which to consistently biopsy tissue sections so that identical locations on oysters can be used to standardize the multiplex PCR method. (PI Comments: The PI 's laboratory recognizes tissue sampling as a limitation and recommends sampling of gill, digestive gland and anus, and is in the process of assessing data relative to conventional testing methods) 4. Test reproducibility of multiplex PCR by running samples with single primer pairs to determine if there are any artifacts of the multiplex PCR. The appearance of spurious bands in some of the multiplexed individuals and those individuals with only dermo primers are cause for concern. There could be some competitive exclusion for binding of the DNA polymerase or a bias towards optimal amplification with some primer pairs over others when all primers are multiplexed together. It appears this should be a standard part of the controls needed for conducting this test. (PI Comments. This work has been conducted and as it was the ground work required to develop and optimize the MPCR methodology. A semi-quantitative MPCR method is in development) 5. Offer training sessions for laboratories interested in using the fully developed test. (PI Comments. The MPCR testing methodology is being modif ed to make the test more user friendly, faster, and a more applicable diagnostic tool to industry and researh. we have expanded the use of the MPCR by utilizing the amplif ed products in a semiquantitative, sensitive, reverse dot blot test.) IMPACTS: We have interesting results in preliminary field trials, which indicate a higher prevalence of SSO in the LIS then previously recorded by conventional histology. We are working with VIMS (Nancy Stokes and CT Bureau of Aquaculture (Inke Sunila) in interpreting and following up on our findings with regards to SSO prevalence and the possibility of new


Haplosproidium strains and/or species. The field trials are expected to be enlightening with regards to the prevalence of Dermo, SSO, and MSX in LIS and the region relative to histology. That is, the multiplex PCR has proven to be much more sensitive (~2X) than conventional methods. RECOMMENDED FOLLOW-UP ACTIVITITES: To develop and optimize a reverse dot-blot assay for the semi-quantification measure of infection by the oyster pathogens MSX, SSO, and Dermo, in the eastern oyster. To make the multiplex PCR a more user friendly, faster, and more applicable diagnostic tool to industry and research, we plan expand the use of the multiplex PCR by utilizing the amplified products in a semi-quantitative, sensitive, reverse dot blot test. PAPERS PRESENTED: • Papers Presented: Soledad Penna, J. Volk, J. Karolus, I. Sunila, and R. A. French. Development and application of multiplex PCR for screening of shellfish pathogens. The 18th Milford Aquaculture Seminar, February 23 25, 1998. R. A. French. Multiplex PCR: A Rapid Diagnostic Method for Screening of Disease Pathogens of Animals and Public Health Significance, Universitv of Illinois, Department of Pathobiologv. ADril 22. 1998 Soledad Penna and R. A. French. Development and application of multiplex PCR for screening of shellfish pathogens. New England Association of Parasitologists (NEAP IV), May 9, 1998. Soledad Penna and R. A. French. Development and application of a multiplex PCR for screening of shellfish pathogens. Universtiv of Connecticut Graduate Research Forum. November 20. 1998. Richard A. French, Salvatore Frasca, Jr., Sylvain De Guise, and Herbert J. Van Kruiningen. Aquatic Animal Health and UCONN Aquaculture Program: New Faculty and Opportunities. The 19th Milford Aquaculture Seminar, February 22-24 1999. Soledad Penna and Richard A. French, John Volk, John Karolus, and Inke Sunila, Roxanna Smolowitz. Diagnostic Screening of Oyster Pathogens: Preliminary Field Trials of Multiplex PCR. 92st

Annual Meeting of the National Shellfisheries Association, Halifax, Nova Scotia, April 18-22, 1999. Spencer Russell, Soledad Penna, Richard French. Comparative evaluation of the multiplex PCR with conventional detection methods for Haplosporidium nelsoni (MSX), Haplosporidium costale (SSO), and Perknsus marinus (Dermo) in the eastern oyster (Crassostrea virginica). The 20th Milford Aquaculture Seminar, February 28 - March 1, 2000. Spencer Russell, Soledad Penna, Richard French. Comparative evaluation of the multiplex PCR with conventional detection methods for Haplosporidium nelsoni (MSX), Haplosporidium costale (SSO), and Perknsus marinus (Dermo) in the eastern oyster (Crassostrea virginica). 93rd Annual Meeting of the National Shellfisheries Association, Seattle, Washington, March 19-23, 2000. Spencer Russell, Soledad Penna, Richard French. Diagnostic Techniques for the Detection of Dermo, MSX, SSO in the Eastern Oyster, Crassotrea virginica. Long Island Sound Research Conference, University of Connecticut, Stamford Campus, Stamford, CT, November 17-18, 2000. S Russell, S Penna, R. A. French. Diagnostic Techniques for the Detection of Dermo, MSX and SSO in The Eastern Oyster, Crassostrea virginica. Proceedings, 2000 Long Island Sound Conference, The Long Island Sound Foundation, Inc., November 2000. • Prospective Conference Presentations National Shellfish Association Annual aeneral meetinz. Mvstic Seasort Connecticut March 2002. MANUSCRIPTS: Maria-Soledad Penna, and Richard A. French. Development of a multiplex PCR for the detection of Haplosporidium nelsoni, Haplosporidium costale, and Perkinsus marinus in the eastern oyster (Crassostrea virginica, Gmelin, 1971) (submitted 03/01, Molecular and Cellular Probes) MANUSCRIPTS IN PREPARATION: 1. "Multiplex PCR field trials for the diagnosis of oyster pathogens MSX, SSO and Dermo: An Atlantic coast perspective." Published in " Diseases of Aquatic Organisms" or "Journal of Shellfish Research""


2. "A Multiplex PCR and reverse dot-blot for the specific non-radioactive detection of MSX, SSO and Dermo in the Eastern oyster, Crassostrea virginica." Published in "Diseases of Aquatic Organisms" of " Molecular and Cellular Probe The Full Report with all the data, graphs and tables is available at the NRAC office upon request.


V. PROJECT PROGRESS REPORTS


PROJECT PROGRESS REPORT

98-5 “Improving intestinal and renal phosphate absorption in fish” Progress Report Period: September 1, 1999 – February 28, 2001 NRAC Total Funding: $179,380 (September 1, 1998 – Augues 31, 2000) (No-Cost Extension through May 31, 2001) Principal Investigators: Ronaldo P. Ferraris, New Jersey Medical School Participating Investigators/ Cooperative Agencies:

Dr. Relicardo Coloso New Jersey Medical Center New Jersey Dr. Nichole McDaniel New Jersey Medical Center New Jersey Dr. Pedrick Weis New Jersey Medical School New Jersey Mr. Mike Hendrix Aquaculture Research Facility, USFWS Pennsylvania Dr. Mark Subramanyam Zeigler Bros., Inc Pennsylvania

PROJECT OBJECTIVES Phosphorus (P) in aquaculture effluents emanates mostly from metabolic wastes and uneaten food. The long-term goal of this project is to reduce total and inorganic P levels in effluents from aquaculture by improving intestinal absorption and reducing renal excretion of phosphates (Pi). Phosphate concentrations in the fecal matter would be reduced by enhancing Pi absorption from the food into the blood, while those in the urine would be reduced by enhancing reabsorption of Pi from the kidney filtrates back into the blood. Phosphorus in aquaculture effluents emanates mostly from metabolic wastes and uneaten food. The long-term goal of this project is to reduce total and inorganic P levels in effluents from aquaculture by improving intestinal absorption and reducing renal excretion of Pi. Phosphate concentrations in the fecal matter would be reduced by enhancing Pi absorption from the food into the blood, while those in the urine would be reduced by enhancing reabsorption of Pi from the kidney filtrates back into the blood. The objectives of the present proposal are: (2.1.1) to screen and refine, in medium-scale feeding trials, diets known to enhance intestinal Pi absorption in the laboratory for their effectiveness in reducing P levels in the soluble, small particulate, and large particulate components of aquaculture effluents; (2.1.2) to evaluate whether practical, industry-type diets formulated based on effective diets from 2.1.1

can reduce P levels in aquaculture effluent and maintain growth rates equal to those of control diets, (2.1.3) to determine the presence of Pi transporters in trout kidney and trout gills, (2.1.4) to assess whether the enzymes phytase and alkaline phosphatase can be regulated by changes in levels of dietary P and dietary vitamin D. (2.1.5) to assess whether renal Pi transporters can be regulated by dietary vitamin D.

ANTICIPATED BENEFITS Retention of P by fish is so poor that even modest increases in intestinal and renal reabsorption can generate significant reductions in levels of urine P. Project results should eventually lead to a reduction in feed-derived P in the effluent, to an increased understanding of the role played by fecal and renal Pi excretion in the generation of effluent P. These results will have a wide-ranging impact on the industry: farms and hatcheries may have a new method for reducing feed-derived P in the effluent while the feed industry can incorporate the findings in feed formulations containing recommended levels of dietary vitamin D and P. The ultimate beneficiary will be the environment. The project’s impact will not be limited to a single sector nor to a single region of the aquaculture economy, as aquaculture effluents with excessive P levels are a problem that abounds in all sectors and locales. The importance and relevance of this study is evident from the support it has received from four of the largest feed manufacturers in the country.


PROGRESS AND PRINCIPAL ACCOMPLISHMENTS Administrative. During most of 1999 and part of 2000, our collaborators at the Northeast Fishery Center had problems with their water supply. This problem has impacted our work and resulted in some delays. Water supply problems have mostly been resolved by June 2000 when we started our practical diet experiment (objective 2.1.2). Ms. Sandra Basantes, a laboratory assistant since 1996 resigned in August 4, 2000 and has been replaced by Ms. Jacqueline Choi. Another student laboratory assistant, Ms. Emilia Gagiu, was hired in December 2000 to help in analyzing the large number of fish, soluble, particulate and fecal samples collected in the practical diet experiment. Manuscript Writing. Two manuscripts based on results from the first two years (1996 - 98) of the project, have been published, and reprints sent to NRAC. For the 1998 - 2001 project, a manuscript has been accepted in Aquaculture, and a preprint is attached. Another manuscript from the 1999 experiment has been completed and final drafts have been distributed to various coauthors for their comments. Completed Experiments. (1 and 2) Effects of dietary P on P concentrations in effluents from the tank culture of juvenile rainbow trout (Phase 1, May to July 1998; Phase 2, November to December 1998) Important discovery. After analyzing the preliminary results of this work, we did a balance equation (ingested P = amount of P in fish growth + amount of P in the soluble component of the effluent + amount of P in the fecal matter), then discovered that we could not account for a significant amount of the effluent P. We therefore hypothesized that the missing P must come from particulates which we missed. Hence, we decided to sample the effluent for particulate P in the next experiment. Galley proofs of accepted manuscript are attached. Completed Experiment. Effects of vitamin D on P concentrations in effluents from the tank culture of juvenile rainbow trout (Phase 3, June - July 1999) Vitamin D3 stimulates small intestinal inorganic phosphate (Pi) absorption and renal Pi reabsorption in mammals, and may also regulate phosphorus (P) homeostasis in fish as well as P levels in effluents from fish culture. A study was conducted to evaluate

the effects of dietary vitamin D3 on reducing the P concentrations in the effluent from tank culture, P utilization, plasma and intestinal luminal Pi concentrations, and concentrations of vitamin D metabolites in the plasma of rainbow trout fed diets already containing low dietary P (0.3 g/ 100 g). Rainbow trout (initial average wt 73 g) were fed semi-purified diets containing 0.3 g P /100 g diet and 625, 2500, 10000, or 40000 IU vitamin D3/ kg diet for 31 d. Fish fed a diet containing 0.6 g P/ 100 g diet and 2500 IU vitamin D3/ kg diet served as control. Plasma Pi concentrations were higher in fish fed 2500 IU/ kg (3.53 + 0.12 mM) similar to those of control fish (Fig. 1). Fish fed 625 or 40000 IU/kg had lower plasma Pi concentrations (2.72 + 0.04 and 2.73 + 0.07 mM, respectively). Plasma concentrations of 25-hydroxyvitamin D (25-(OH)D) and 1,25-dihydroxyvitamin D (1,25-(OH)2D) were unaffected by dietary vitamin D3 levels. Increasing levels of dietary vitamin D3 also did not alter the Pi concentrations in the intestinal lumen. As previously observed, dietary P is a major factor affecting soluble and fecal P concentrations in the effluent. In this study, soluble P concentrations sampled at the peak of soluble P excretion (at 1430 H) were significantly lower in fish fed 2500 IU/ kg or higher than in those fed 625 IU/ kg and the control diet. Soluble P concentrations in the effluent increased after each feeding and returned to baseline levels in the early morning hours (Figs. 2,3). In fecal dry matter, fish fed 2500 IU/ kg had significantly lower P levels than those fed 40000 IU/ kg and the control diet (Fig. 4). Particulate P concentrations increased in the early afternoon and into the evening hours, but were unaffected by dietary vitamin D3 (Fig. 5). Growth, survival, and carcass P levels were also unaltered by dietary vitamin D3. P utilization was unaffected by dietary vitamin D3 over the feeding period. Thus, in trout fed low P diets, dietary vitamin D3 at 2500 IU/kg increases plasma Pi concentrations, attenuates the increases in soluble P concentrations during the peak excretion time, and decreases fecal P content. Dietary vitamin D3 had no effect on fish growth and survival, as well as Pi concentrations in the intestinal lumen, circulating vitamin D metabolites, and P utilization over the feeding period in this study. Ongoing Experiment. Dietary regulation of intestinal and renal Pi transporter mRNA and protein in juvenile rainbow trout (experiment on October 1999 - February 2000, tissue analysis ongoing)


We conducted an experiment using three semipurified diets (0.6 % P, 2500 IU/kg vitamin D; 0.6% P, 10000 IU/kg vitamin D and 0.12% P, 10000 IU/kg vitamin D) and fed the trout these diets for 30 and 60 days, to determine whether dietary P or vitamin D affect the expression NapiII protein and mRNA. Renal, intestinal, gill and caecal tissues were collected. Preliminary results from Northern blot analysis indicate that dietary P affects the level of NaPiII mRNA in trout intestine and kidney. Preliminary results from immunocytochemistry revealed that NaPiII protein are indeed found in trout intestinal cells, at the brushborder membrane, as well as in trout kidney cells, at the basolateral membrane. Ongoing analyses are being done, using additional samples. Ongoing Experiment. Evaluation of practical, industry-type diets formulated based on effective diets from 2.1.1 in a long-term feeding experiment (Phase 4, actual field work, May 2000 - September 2000; laboratory analysis of samples, ongoing) We have now designed practical diets using low P formulations, and intend to determine how trout grow in these diets in long term feeding experiments, and how effluent P partitioning is affected. Practical diets used feed ingredients similar to industry (fish meal, blood protein, feather meal, etc.). Trout were fed practical diets as follows: Diet 1, 0.6 % P, 2500 IU/kg vitamin D (control); Diet 2, 0.6 %P, 10000 IU/kg; Diet 3, 0.9% P, 2500 IU/kg; Diet 4, 1.2 %P, 2500 IU/kg and Diet 5, trout chow. Soluble P, particulate P, and fecal P were sampled as in Phase 3. Growth of trout was greatest in Diet 5, but weight in Diet 5 was only 6% greater than diet 4, which in turn was 12% greater than Diets 1 – 3 (Fig. 6). Percent biomass gained and feed efficiency ratios were also greater in Diets 4 and 5 (Fig. 7 & 8). In contrast, feed conversion ratios were greatest in Diet 3 (Fig. 9). Analysis of fish tissues and effluent samples is ongoing. Soluble P analysis is almost done (Fig. 10), showing again that effluents from trout fed diets with high P levels also have high soluble P concentrations. The main difference between these practical diets and the semi purified diets used in Phase 1 – 3 is that semipurified diets yield high peaks of soluble effluent P right after feeding.

WORK PLANNED March 2001 • Analysis of particulate samples from September

2000 • Analysis of fecal samples from September 2000 • Analysis of initial carcass samples from July

2000 • Analysis of carcass samples from September

2000 • Immunocytochemistry of tissues from September

2000 • Northern blot analysis of tissues from

February 2000 • Northern blot analysis of tissues from September

2000 • Statistical analysis of soluble P • Design diets and plan for effluent experiment for

Summer 2001 April 2001 • Finalize manuscript from Phase 3 (1999 work) • Northern blot analysis of tissues from September

2000 • Analysis of carcass P samples from September


2000 • Statistical analysis of Northern blot results from

February 2000 • Statistical analysis of plasma Pi and fecal P from

Phase 4 • Request for no cost extension • Set-up effluent experiment May 2001 • Submit manuscript from Phase 3 (1999 work) • Statistical analysis of particulate P from

September 2000 • Prepare manuscript for laboratory experiment on

January and February 2000 • Northern blot analysis of tissues from September


2000 • Conduct experiments, collect samples (soluble,

particulate, fecal, carcass, plasma) June 2001 • Statistical analysis of carcass P from September

2000 • Conduct field experiments and collect samples


• Analyze soluble P from effluents of May 2001 experiment

• Continue manuscript of laboratory experiment on January and February 2000

July 2001 • Statistical analysis of Northern blots of tissues

from September 2000 • Conduct field experiment and collect samples • Analyze soluble P from effluents of June 2001

experiment • Finalize manuscript of laboratory experiment on

January and February 2000

August 2001 • Conduct field experiment and collect samples • Analyze fecal P from May – June 2001 • Submit manuscript of laboratory experiment on

January and February 2000

IMPACTS This project offers a novel approach towards a solution by proposing to enhance intestinal Pi absorption using dietary components already present in fish diets. Information from results will lead to reduced phosphorus content in aquaculture effluents and to increased understanding of the role played by intestinal and renal Pi absorption in the generation of feed-derived pollutants. PUBLICATIONS, MANUSCRIPTS Avila, E.M., S.P. Basantes and R.P. Ferraris. 1999.

Cholecalciferol modulates plasma phosphate but not plasma vitamin D levels and intestinal phosphate absorption in rainbow trout (Oncorhynchus mykiss). Gen. Comp. Endocrinol. 114:460-469.

Avila, E.M., , H. Tu, S.P. Basantes and R.P. Ferraris. 2000. Dietary phosphorus regulates intestinal transport and plasma concentrations of phosphate in rainbow trout. J. Comp. Physiol. B. 170:201-209.

Coloso, R.M., S.P. Basantes, K. King, M.A. Hendrix, J.W. Fletcher, P. Weis and R. P. Ferraris. Effect of dietary P and vitamin D on soluble phosphate levels in effluents from the experimental culture of rainbow trout. Aquaculture (in press).

PAPERS PRESENTED Coloso, R.M., E.M. Avila, S.P. Basantes and R.P.

Ferraris. Influence of dietary phosphorus and vitamin D on intestinal phosphate uptake, plasma and luminal phosphate concentrations, and alkaline phosphatase and phytase activities in rainbow trout. World Aquaculture Society, New Orleans, LA Feb. 2-7, 2000.

Ferraris, R.P., R.M. Coloso, S.P. Basantes, K. King, J. Fletcher, M. Hendrix and P. Weis. Soluble phosphate concentrations in effluents from experimental trout culture: influence of dietary phosphorus and vitamin D, feeding activity and circadian rhythm. World Aquaculture Society, New Orleans, LA Feb. 2-7, 2000.

Ferraris, R.P., R.M. Coloso, S.P. Basantes, K. King, J. Fletcher, M. Hendrix and P. Weis. Partitioning of phosphates in effluents from experimental trout culture: the phantom menace is soluble and suspended. World Aquaculture Society, New Orleans, LA Feb. 2-7, 2000.

Coloso, R.M., S.P. Basantes, A. Werner and R. P. Ferraris. Effect of dietary P on sodium-phosphate ransporter expression in rainbow trout intestine and kidney. FASEB, March 31 – April 4, 2001, Orlando, FL.




98-5 “Improving Intestinal and Renal Phosphate Absorption in Fish” Progress Report Period: September 1, 1999 - August 31, 2001 NRAC Total Funding: $179,380 (September 1, 1998 – Augues 31, 2000) (No-Cost Extension through May 31, 2001) Principal Investigator: Ronaldo P. Ferraris, New Jersey Medical School Participating Investigators/ Cooperative Agencies:

Dr. Relicardo Coloso New Jersey Medical Center New Jersey Dr. Nichole McDaniel New Jersey Medical Center New Jersey Dr. Pedrick Weis New Jersey Medical School New Jersey Mr. Mike Hendrix Aquaculture Research Facility, USFWS Pennsylvania Dr. Mark Subramanyam Zeigler Bros., Inc Pennsylvania

PROJECT OBJECTIVES Phosphorus (P) in aquaculture effluents emanates mostly from metabolic wastes and uneaten food. The long-term goal of this project is to reduce total and inorganic P levels in effluents from aquaculture by improving intestinal absorption and reducing renal excretion of phosphates (Pi). Phosphate concentrations in the fecal matter would be reduced by enhancing Pi absorption from the food into the blood, while those in the urine would be reduced by enhancing reabsorption of Pi from the kidney filtrates back into the blood. Phosphorus in aquaculture effluents emanates mostly from metabolic wastes and uneaten food. The long-term goal of this project is to reduce total and inorganic P levels in effluents from aquaculture by improving intestinal absorption and reducing renal excretion of Pi. Phosphate concentrations in the fecal matter would be reduced by enhancing Pi absorption from the food into the blood, while those in the urine would be reduced by enhancing reabsorption of Pi from the kidney filtrates back into the blood. The objectives of the present proposal are: (2.1.1) to screen and refine, in medium-scale feeding trials, diets known to enhance intestinal Pi absorption in the laboratory for their effectiveness in reducing P levels in the soluble, small particulate, and large particulate components of aquaculture effluents; (2.1.2) to evaluate whether practical, industry-type diets formulated based on effective diets from 2.1.1

can reduce P levels in aquaculture effluent and maintain growth rates equal to those of control diets, (2.1.3) to determine the presence of Pi transporters in trout kidney and trout gills, (2.1.4) to assess whether the enzymes phytase and alkaline phosphatase can be regulated by changes in levels of dietary P and dietary vitamin D. (2.1.5) to assess whether renal Pi transporters can be regulated by dietary vitamin D. ANTICIPATED BENEFITS Retention of P by fish is so poor that even modest increases in intestinal and renal reabsorption can generate significant reductions in levels of urine P. Project results should eventually lead to a reduction in feed-derived P in the effluent, to an increased understanding of the role played by fecal and renal Pi excretion in the generation of effluent P. These results will have a wide-ranging impact on the industry: farms and hatcheries may have a new method for reducing feed-derived P in the effluent while the feed industry can incorporate the findings in feed formulations containing recommended levels of dietary vitamin D and P. The ultimate beneficiary will be the environment. The project’s impact will not be limited to a single sector nor to a single region of the aquaculture economy, as aquaculture effluents with excessive P levels are a problem that abounds in all sectors and locales. The importance and relevance of this study is evident from the support it has received from four of the largest feed manufacturers in the country.


PROGRESS AND PRINCIPAL ACCOMPLISHMENTS Administrative. An experiment utilizing practical diets and monitoring growth for several months was conducted from July to November 2000, and samples analyzed during the year. There were four practical diets and one commercial diet. Because of the large number of samples collected for effluent analysis, a second student assistant, Ms. Emilia Gagiu, was hired from November 2000 to July 2001 to help with sample processing. In November 2000, the principal investigator received a three year USDA - National Research Initiative grant based on work supported by NRAC. In September 2001, the postdoctoral fellow, Dr. Relicardo Coloso, will leave the project and will be returning to his home country after three years of service to this project. He will finish the manuscripts supported by funds from this project. Dr. Shozo Sugiura, an expert of P metabolism in fish, has been hired to replace him. Dr. Sugiura will be supported by the new USDA grant. Manuscript Writing. Two manuscripts based on results from the first two years (1996 - 98) of the project, have now been published. A third manuscript is in press in Aquaculture (a preprint is attached). A fourth manuscript has been submitted and is undergoing revision. A fifth manuscript is nearing completion. A total of 5 - 6 manuscripts will have arisen from the five years of NRAC support. An extension agent has been contacted to help in the publication of a fact sheet after the above manuscripts have been accepted. Completed Field Experiment. Effects of dietary P on P concentrations in effluents from the tank culture of juvenile rainbow trout (Phase 2 , November to December 1998) Results from this work has been reported in detail in the annual report submitted August 1999, and is summarized in the Aquaculture preprint. Completed Field Experiment. Effects of vitamin D on P concentrations in effluents from the tank culture of juvenile rainbow trout (Phase 3, June - July 1999) Results from this work has been reported in detail in the annual report submitted August 2000, and is described at length in the Aquaculture preprint. The most important result from this initial work is the fact that we can account for the amount of P fed to fish, and partition that P into amount of P gained by fish + amount of P in the fecal matter + amount of P

in the particulate matter + amount of P in the soluble component. Completed Indoor Experiment. Dietary regulation of intestinal and renal Pi transporter mRNA and protein in juvenile rainbow trout (October 1999 - February 2000) In the 1996-98 phase of this project, we were unable to consistently detect the NaPiII mRNA in trout. An expert on NaPiII in England helped us design a probe that could detect trout NaPiII mRNA in the intestine and kidney. These probes were tested in our laboratory in October and November 1999 and we were able to detect NaPiII mRNA in trout intestine and kidney. We were also able to design antibodies (raised in rabbits by Sigma) against the NaPi2 protein, for determining NaPiII cellular location in trout intestine and kidney using immunocytochemistry. We then conducted an experiment using three semipurified diets (0.6 % P, 2500 IU/kg vitamin D; 0.6% P, 10000 IU/kg vitamin D and 0.12% P, 10000 IU/kg vitamin D) and fed the trout these diets for 30 and 60 days, to determine whether dietary P or vitamin D affect the expression NapiII protein and mRNA. Renal, intestinal, gill and caecal tissues were collected. Like results previously observed in mammals, Northern blot analysis indicate that dietary P affects the level of NaPiII mRNA in trout intestine and kidney (Figs. 1 and 2). Preliminary results from immunocytochemistry revealed that NaPiII protein are indeed found in trout intestinal cells, at the brushborder membrane, as well as in trout kidney cells, at the basolateral membrane. Ongoing analysis are being done, using additional samples. Completed Field Experiment. Evaluation of practical, industry-type diets formulated based on effective diets from 2.1.1 in a long-term feeding experiment (Phase 4, actual field work, May 2000 - September 2000; laboratory analysis of samples, October 2000 - July 2001) We designed practical diets using low P formulations, and intend to determine how trout grow in these diets in long term feeding experiments, and how effluent P partitioning is affected. Practical diets used feed ingredients similar to industry (fish meal, blood protein, feather meal, etc.). Trout were fed practical diets as follows: Diet 1, 0.6 % P, 2500 IU/kg vitamin D (control); Diet 2, 0.6 %P, 10000 IU/kg; Diet 3, 0.9% P, 2500 IU/kg; Diet 4, 1.2 %P, 2500 IU/kg and Diet 5, trout chow. Soluble P,


particulate P, and fecal P were sampled as in Phase 3. Like soluble P, particulate P involved hourly samples, over 24 h, of the effluent. Two series of 24 h samples are done: one near the beginning of the experiment, another towards the end. Trout fed these practical diets grew by about 250% over an 11 week period (Fig. 3, please see Appendix 1 below). Fig. 4 shows a 24 h profile of soluble P in effluents collected from the tanks near the end of the experiment in September 2000. Results clearly show that at the density (~25 kgbiomass/0.4 m3) and flow rate (12 l/min), the concentrations of soluble P in the effluent exceeds the limits (0.01 to 0.06 ppm or mg/l) allowed in Pennsylavania and New Jersey. The average daily soluble P in September 2000 is summarized in Figure 5, while that in July 2000 is summarized in Figure 6. In both cases, the concentration of soluble P in the effluent increases with dietary P. Note that when fish density and fish size is low, as would be the case in July 2000, soluble P concentrations in the effluent are also low. Fig. 7 depicts a 24 h profile of particulate P content (sampled by filtering the effluent with a 10 �m mesh filter), and Fig. 8 is the average daily particulate P in September 2000. Results indicate that particulate P contributes a significant amount of P in the effluent. Particulate P concentrations also do not change with fish size and biomass, as indicated by the similarity of the average daily particulate P in July (Fig. 9) and September 2000. Fig. 10 depicts the average P content of fecal matter collected from the tanks in July 2000. P in fecal matter is much higher in commercially-obtained trout chow. Similar observations were obtained in September 2000 (Fig. 11). The P content in fish carcass is summarized in Fig. 12 (percent ash) and Fig. 13 (percent of dry weight). These values are well within those reported in the literature. The P content in the ash does not differ from that of the initial (22 + 2 %), but the P content in the dry matter (2.36 + 0.19 %) of initial fish was significantly greater than those of fish sampled at the end of the experiment (Fig. 13). Our most important findings are summarized as preliminary results of a phosphorus balance equation, where we account for total P intake, now presented as Fig. 14. In July 2000, three weeks after the start of the experiment, most of the P consumed by the fish become incorporated as flesh (~50% or more). In fish fed P-sufficient diets (0.6%), most of the P in the effluent are found in the fecal portion, and some in the particulate portion. When fish are shifted to a P-excess diet (0.9% and 1.2%), most of the P in the

effluent shifts to the soluble component. We are now currently calculating the results for the November 2000 analyses. WORK PLANNED September to December 2001 Statistical analysis of carcass P Calculate balance equation for September 2000

experiment Completion of manuscript from Phase IV Revision of manuscript from Phase III Completion of manuscript from the indoor

experiment END OF PROJECT IMPACTS This project offers a novel approach towards a solution by proposing to enhance intestinal Pi absorption using dietary components already present in fish diets. Information from results will lead to reduced phosphorus content in aquaculture effluents and to increased understanding of the role played by intestinal and renal Pi absorption in the generation of feed-derived pollutants.

PUBLICATIONS, MANUSCRIPTS Avila, E.M., S.P. Basantes and R.P. Ferraris. 1999.

Cholecalciferol modulates plasma phosphate but not plasma vitamin D levels and intestinal phosphate absorption in rainbow trout (Oncorhynchus mykiss). Gen. Comp. Endocrinol. 114:460-469.

Avila, E.M., H. Tu, S.P. Basantes and R.P. Ferraris.

2000. Dietary phosphorus regulates intestinal transport and plasma concentrations of phosphate in rainbow trout. J. Comp. Physiol. B. 170:201-209.

Coloso, R.M., S.P. Basantes, K. King, M.A. Hendrix,

J.W. Fletcher, P. Weis and R.P. Ferraris. 2001. Effect of dietary P and vitamin D on soluble phosphate levels in effluents from the experimental culture of rainbow trout. Aquaculture (in press).

Coloso, R.M., K. King, J.W. Fletcher, M.

Subramanyan, M.A. Hendrix, P. Weis and R.P. Ferraris. In preparation. Phosphorus utilization in rainbow trout (Oncorhynchus mykiss) fed


practical diets and its consequences on effluent phosphorus levels.

PAPERS PRESENTED Coloso, R.M., E.M. Avila, S.P. Basantes and R.P.

Ferraris. Influence of dietary phosphorus and vitamin D on intestinal phosphate uptake, plasma and luminal phosphate concentrations, and alkaline phosphatase and phytase activities in rainbow trout. World Aquaculture Society, New Orleans, LA Feb. 2-7, 2000.

Ferraris, R.P., R.M. Coloso, S.P. Basantes, K. King,

J. Fletcher, M. Hendrix and P. Weis. Soluble phosphate concentrations in effluents from experimental trout culture: influence of dietary phosphorus and vitamin D, feeding activity and circadian rhythm. World Aquaculture Society, New Orleans, LA Feb. 2-7, 2000.

Ferraris, R.P., R.M. Coloso, S.P. Basantes, K. King,

J. Fletcher, M. Hendrix and P. Weis. Partitioning of phosphates in effluents from experimental trout culture: the phantom menace is soluble and suspended. World Aquaculture Society, New Orleans, LA Feb. 2-7, 2000.

Coloso, R.M., S.P. Basantes, A. Werner and R.P.

Ferraris. Effect of dietary phosphorus on sodium-phosphate transporter expression in trout intestine and kidney. FASEB J. 15(5):A841, 2001.




98-7 “Aquaculture Curricula Resource Guide Publication and Distribution” Progress Report Period: February 11, 1999 – February 17, 2000 NRAC Total Funding: $10,000 (February 1, 1999 – December 31, 2000) Principal Investigator: Scott Soares, Massachusetts Department of Agriculture Participating Investigators/ Cooperative Agencies:

Fenna Hanes NEBHE Massachusetts Clare Lewis NEBHE Massachusetts Dale Leavitt SEMAC, WHOI Sea Grant Massachusetts Joe Buttner Salem State College Massachusetts Richard Audet Roger Williams University Rhode Island

PROJECT OBJECTIVES: i. Organize an NRAC advisory group/committee

for format and content suggestions, review and critique of the Aquaculture Curricula Resource Guide.

ii. Identify, review and summarize existing national and international aquaculture curricula that have been developed for elementary, secondary, vocational technical and vocational agricultural schools.

iii. Synthesize curricula summaries and catalog according to target audience, content, source and utility.

iv. Develop and issue a request for bids that is consistent with the procurement laws and policies of the Commonwealth of Massachusetts for the electronic and hard copy publication and broad distribution of the Aquaculture Curricula Resource Guide.

v. Formulate an agreement between the Massachusetts Department of Food and Agriculture, the New England Board of Higher Education and the Northeastern Regional Aquaculture Center regarding the ownership, distribution and repository of the master Aquaculture Curricula Resource Guide.

vi. Provide educators of various grade levels throughout the Northeastern United States and beyond a comprehensive aquaculture curriculum resource guide that will facilitate and enhance aquaculture education efforts.

vii. Increase public awareness and enhance aquaculture educational endeavors, thereby improving conditions for long term development of the aquatic cultivation industry of the Northeast and abroad.

ANTICIPATED BENEFITS: It is the intent of this project to facilitate educator and student access to aquaculture curricula and aquaculture education materials. A number of elementary, secondary and post-secondary education programs have demonstrated that educational programs that use aquaculture themes or systems have great impact on student enthusiasm and willingness to cooperate in classroom activities. Whether teaching about aquaculture or teaching with aquaculture, programs that employ aquaculture in the classroom also provide the opportunity for exposing students to the great variety of aquaculture species and culture techniques. Through such exposure, students and their parents can achieve a better understanding of what aquaculture is and the potential that aquaculture may hold. In many States, the expansion of the aquaculture industry relies on the willingness of the local municipalities to approve project development. Accordingly, increased opportunities for public education can promote a better understanding of what aquaculture is thereby increasing public awareness and providing opportunities for better informed decision making.


PROGRESS AND PRINCIPAL ACCOMPLISHMENTS: i. Organize an NRAC advisory group/committee

for format and content suggestions, review and critique of the Aquaculture Curricula Resource Guide.

A draft copy of the Aquaculture Curricula Resource Guide (Attachment A) has been sent to the following individuals with a request for comments and review assistance: Dr. Susan Case, Biology Department Chair, Salem State College, Mr. Bill Clark, Director Cape Cod Cooperative Extension, Mr. John Ewart, University of Delaware, Dr. Gary Jensen, CSREES USDA, Commissioner Jonathan Healy, MA Department of Food and Agriculture, Mr. Jack Hoy, President New England Board of Higher Education, Attny. Lawrence McCormick, Chief Council MA Department of Food and Agriculture, Dr. Judy McDowell, Director Woods Hole Oceanographic Institution Sea Grant Program, Dr. James Mcvey, Sea Grant National Program Leader, DOC, Dr. Lance Stewart, UCONN Cooperative Extension Service and Dr. Bill Wise Chair Technical Industry Advisory Committee NRAC. Although the individuals requested to review the Guide includes representation beyond NRAC, as a result of the numerous organizations that participated in this project the review committee was expanded from the original proposal in effort to provide the opportunity for comment and review from each of the organizations that were represented by the project participants.

ii. Identify, review and summarize existing national and international aquaculture curricula that have been developed for elementary, secondary, vocational technical and vocational agricultural schools.

Information about the process and methods for identification, review and summary development was presented in the first progress report for this project (February 11, 1998 – February 17, 1999). The aquaculture curricula identified and the review summaries (i.e. aquaculture curricula profiles) are presented in Attachment A of this report (Draft Aquaculture Curricula Resource Guide).

iii. Synthesize curricula summaries and catalog according to target audience, content, source and utility.

The aquaculture curricula identified and the review summaries (i.e. aquaculture curricula profiles) are presented in Attachment A of this report (Draft Aquaculture Curricula Resource Guide).

iv. Develop and issue a request for bids that is

consistent with the procurement laws and policies of the Commonwealth of Massachusetts for the electronic and hard copy publication and broad distribution of the Aquaculture Curricula Resource Guide.

Since the development of the proposal for this project, the MA Department of Food and Agriculture has hired a graphic artist/publication specialist. A final draft of the Aquaculture Curricula Resource Guide will be developed as completion of the curricula review is approached. The final draft of the Aquaculture Curricula Resource Guide will be used as part of the solicitation for a vendor that will provide electronic and hard copy publication services. Although names and addresses for eventual mailing of the completed Guide have been compiled, no work has been done toward the development of a request for bids.

v. Formulate an agreement between the

Massachusetts Department of Food and Agriculture, the New England Board of Higher Education and the Northeastern Regional Aquaculture Center regarding the ownership, distribution and repository of the master Aquaculture Curricula Resource Guide.

No work has been done toward the completion of this objective.

vi. Provide educators of various grade levels

throughout the Northeastern United States and beyond a comprehensive aquaculture curriculum resource guide that will facilitate and enhance aquaculture education efforts.

An abridged list of curricula resources and educational materials was prepared and provided to participants of the NEBHE AQUA project November 1997 meeting of interested project


participants. 112 copies of the material were distributed. Those materials and the resources collected through the survey were compiled into a working list (Attachment C) from which the selected curricula were taken. The comprehensive resource guide will be generated through the analysis of the selected curricula and will be the final product of this project.

vii. Increase public awareness and enhance

aquaculture educational endeavors, thereby improving conditions for long term development of the aquatic cultivation industry of the Northeast and abroad.

Although the final impact and benefit resulting from the distribution of the Aquaculture Curricula Resource Guide will not be achieved until the completion of this project, increased awareness and enhancement of educational programs has been a result of frequent interaction between the project participants and the public/education institutions. Exposure to previously unknown educational materials has enable project participants to achieve a greater awareness of the availability of aquaculture educational resources thereby enabling greater dissemination of the information to a broad audience of educators and the general public.

WORK PLANNED:

As a result of reassignments and employment changes, there has been some modification to project participants. To that end, Dr. Ann Whitney-Roper and Mr. Rob Garrison have not been able to participate with this project. Recent employment changes have also resulted in the inability of Ms. Clare Cookson Lewis to continue working with the project. Dr. Joe Buttner and Dr. Richard Audet have been added as project participants and are serving in the capacity that Mr. Garrison and Dr. Whitney-Roper had previously served. Accordingly, the changes in participants have not negatively impacted the project progress. Delays in receipt of curricula that were identified for review and of volumes of information that are larger than anticipated at the time of proposal development have lead to the need to request a no cost extension from NRAC. During the extension period, curriculum review will be

finalized as will all outstanding objectives (specifically objectives iv - vii).

IMPACTS: Although the greatest impact and benefit from this project to the aquaculture industry will likely occur after the publication and distribution of the Aquaculture Curricula Resource Guide. And although that benefit will perhaps be more long than near term, there has been some immediate indirect benefit that will surely continue in the present. These indirect benefits to the aquaculture industry includes: • Increased public awareness through

teacher/student/parent interaction that will facilitate near term industry expansion

• Creation of a potential workforce for the aquaculture industry when it is able to absorb volumes of trained individuals

• Cultivation of an electorate that has familiarity with aquatic cultivation enabling well informed voting and decision making with regard to aquaculture industry development

PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED: Papers Presented: Soares, Scott J. Aquaculture Curricula Resource

Guide Publication and Distribution Project. Northeastern Regional Aquaculture Center Annual Regional Extension Project meeting. 1998 Boston Fish Exposition. World Trade Center, South Boston, MA. 1998




00-2 “Improving Larval Survival for Black Sea Bass Aquaculture” Progress Report Period: April 4, 1999 – May 15, 2001 NRAC Total Funding: $132,426 (April 4, 1999 – March 31, 2001) (No-Cost Extension through December 31, 2001) Principal Investigator: Terence M. Bradley, University of Rhode Island Participating Investigators/ Cooperative Agencies:

David Berlinsky University of Rhode Island Rhode Island George Flimlin New Jersey Sea Grant New Jersey George Nardi Great Bay Aquafarms New Hampshire Theodore Smith SC Dept. of Natural Resources South Carolina Mark Subramanyam Zeigler Bros, Inc. Pennsylvania Gloria Seaborn DOC/NOAA/NOS South Carolina

PROJECT OBJECTIVES: Presently, survival of pre-feeding larvae is low (<10%), limiting the availability of fish for production and further research and development. To address this major constraint to production we are investigating the cause of these mortalities and seek to develop remedial action. The specific objectives of this study are to: 1. Determine whether low larval survival is related to the time of hormonal induction and spawning. 2. Assess the effect of temperature on fertilization and larval survival. 3. Investigate whether low survival is related to broodstock nutrition. 4. Determine the conditions of environmental salinity and aeration that promote maximum survival of pre-feeding larvae. ANTICIPATED BENEFITS: Despite the increasing demand for seafood and the wealth of endemic marine finfish species in the Northeast, only Atlantic salmon (Salmo salar) and summer flounder (Paralichthys dentatus) are currently cultured on a large commercial scale. The health and growth of the fledgling marine finfish aquaculture industry in the Northeast is dependent on

diversification into culture of other species. Diversification reduces the potential for industry-wide catastrophic disasters such as those caused by species-specific pathogens, market shifts, overproduction and declines in farm gate prices. The marine finfish aquaculture industries in Europe and Asia have embraced this strategy with great success. In Europe, at least 6 species of marine finfish are cultured commercially including salmon, turbot, sea bream, European sea bass and wolfish. Over 20 species, ranging from puffers and yellowtail to bream and groupers, are currently produced on a commercial scale in Japan. The endemic black sea bass is an excellent candidate for providing much needed diversification to the aquaculture industry in the Northeast. Importation and culture of non-native species for aquaculture such as the European sea bass (Dicentrarchus labrax) is unlikely due to state and federal regulations. Initial estimates of growth rates suggest that a market size black sea bass (approximately 500 g) could be produced in some 12 – 14 months. This project will address one of the major impediments to successful culture of black sea bass- poor survival of post hatch larvae. PROGRESS AND PRINCIPAL ACCOMPLISHMENTS: During the current reporting period (October 15, 2000 – May 15 2001) we have focused efforts on investigations related to Objectives 1 and 4. The


following text provides a report of the research conducted during this period: Objective 1. Determine whether low larval survival is related to the time of hormonal induction and spawning. As described in the previous report, we investigated the effects of different photoperiod regimens on reproductive development and spawning advancement. Specifically, 32 females and 12 males were stocked into each of two 2 meter diameter circular, polyethylene tanks supplied with continuously flowing filtered seawater. Two photoperiod cycles were used in the study: a 12-month control cycle simulating natural conditions and a 6-month experimental cycle in which the annual photoperiod cycle was compressed into 6 months. Stage of reproduction in males was assessed by checking for spermiation. In females, oocyte development was monitored by histological examination of ovarian biopsies. Reproductive development in these fish also was tracked by creating a profile of plasma levels of 17B-estradiol (E2), testosterone (T) and 11-ketotestosterone (11-KT). Plasma levels of E2, T and 11-KT were determined by radioimmunoassay (RIA). Analysis of these measures indicates that in males, photothermal manipulation appeared to have limited effect on the timing of reproduction. Males reared under the 6 month compressed cycle and those reared under a natural photoperiod both began spermiating in January and continued until the end of the sampling period. In contrast, in females the timing of follicle development and ovulation was advanced by some two months when exposed to the compressed photoperiod cycle. Compression of the photoperiod also successfully stimulated the advancement of the profile of plasma sex steroids, specifically, 17β-estradiol, testosterone and 11-ketotesterone. Males and females reared under the compressed photoperiod were spawned successfully two months in advance of fish on natural period providing evidence that the time of spawning of black sea bass can be altered by photothermal manipulation. The use of photoperiod to spawn black sea bass at multiple points in the year will provide growers with an inexpensive and practical tool to increase production of larvae. Objective 4. Determine the conditions of environmental salinity and aeration that promote maximum survival of pre-feeding larvae.

Additional experiments were conducted to investigate the effects of environmental salinity on larval survival. Results from the previous experiments indicate that salinities of at least 10 ppt are required for survival of black sea bass larvae. The experiments were repeated with larger numbers of replicates and confirm the previous results. Combined these studies indicate that black sea bass larvae require at least 10 ppt salinity and become more tolerant to lower environmental salinities with development. Data Analysis and Manuscript Preparation: At this time we have begun to compile data from the studies conducted to date for analysis and preparation of the final report and manuscripts. WORK PLANNED: No significant changes have been made to the work plan. Work will continue on all objectives to attain the stated goals of the project. IMPACTS: To date we have made significant progress toward solving the problem of low larval survival that is impeding commercial culture of black sea bass. We are confident that upon completion of the project we will have the information required to alleviate this problem. PUBLICATIONS, MANUSCRIPTS OR PAPERS PRESENTED: Papers Presented Howell, R., D. Berlinsky and T. Bradley. Control of

Reproduction of Black Sea Bass. Annual Milford Aquaculture Workshop. Milford, CT.

February 2001. Journal of Shellfish Research (in press).

D. Berlinsky, R. Howell and T. Bradley. Effect of

Environmental Salinity on Survival and Growth of Early Life Stages of Black Sea Bass. Annual Milford Aquaculture Workshop, Milford, CT. February 2001. Journal of Shellfish Research (in press).




00-3 “Surveillance of Infectious Salmon Anemia Virus (ISAV) in the Northeast” Progress Report Period: August 1, 2000–February 1, 2001 NRAC Total Funding: $45,950 (August 1, 2000 – July 31, 2003) Principal Investigator: John T. Singer, University of Maine Participating Investigators/ Cooperative Agencies:

Eric D. Anderson University of Maine Maine Carol H. Kim University of Maine Maine Deborah A. Bouchard Micro Technologies, Inc. Maine William R. Keleher, Jr. Micro Technologies, Inc. Maine Bruce L. Nicholson University of Maine Maine Paul R. Waterstrat Maine Department of Marine Resources Maine Michael Opitz University of Maine Maine Sharon A. MacLean NOAA NMFS NEFSC Rhode Island

PROJECT OBJECTIVES: Objective 1: Develop and optimize reverse-transcriptase polymerase chain reaction (RT-PCR) assays for the detection and identification of infectious salmon anemia virus (ISAV). Objective 2: Conduct surveillance program for ISAV in Atlantic salmon aquaculture sites in the State of Maine that are at high risk of becoming ISAV-positive. ANTICIPATED BENEFITS: We are using a progressive approach to aid in the avoidance, control, and containment of ISAV in the Northeast. This will be accomplished by having available and using preemptively, existing-, new-, and improved technologies. Our work will contribute to: (1) Development of several PCR assays as rapid and sensitive diagnostic assays for ISAV, (2) Development and implementation of methods for molecular epidemiology to initiate a pilot program to definitively track the transmission and identify possible reservoirs for the dissemination of ISAV, and (3) Development of an integrated, comprehensive surveillance and diagnostic program for ISAV. These technologies will be used by diagnostic, veterinary, vaccine, biotechnology, and academic laboratories. The salmon aquaculture industry will benefit by maintaining good fish health status (ISAV free fish

stocks) and having information available for informed decision making if the virus is diagnosed at a site in Maine. The salmon aquaculture industry will benefit economically from the technologies by help in avoiding and containing ISAV. In a worst-case scenario, the detection of ISAV at a grow-out site, the possible quarantine or destruction of fish and fallowing of production sites would be based on accurate information gathered through diagnosis and molecular epidemiology. This information hopefully will limit the extent of such extreme measures. PROGRESS AND PRINCIPAL ACCOMPLISHMENTS: Anderson Group: We have generated ISAV cDNA libraries composed of 768 pGEM/cDNA plasmid clones. The individual plasmids have been grouped, by nucleic acid hybridization, into ten distinct genetic groups that represent the entire ISAV viral genome. Three of the genetic groups have been found to correspond to ISAV viral segments 2, 5, and 8. The remaining groups are being "screened" by nucleic acid sequencing and Northern blot analysis. We anticipate that March 15, 2001 will mark the completion of this portion of the outlined work. Nicholson Group: A new nested RT-PCR assay for detection and identification of ISAV was developed based on sequences of viral genome segment 2 which encodes the putative polymerase protein. Preliminary results indicate that this new RT-PCT assay is more


sensitive than our previously developed RT-PCR assay. Also, we have developed a RT-PCR assay for ISAV based on recent sequence data for genome segment 7 which encodes the viral matrix (M) protein. MicroTechnologies: Micro Technologies has received and continues to receive a fair volume of diagnostic samples for ISAV screening. Because of this, there was an immediate need for a direct tissue RT-PCR assay to be developed and Micro Technologies developed a direct tissue RT-PCR technique using the primer set established by Bruce Nicholson (really Sharon Blake) that worked well for ISAV diagnostics. We participate in the Canadian QA/QC program and find that our results are very consistent and accurate. Over the last year, however, we have begun working on optimization of the direct tissue procedure and the RT-PCR in general. Essentially, we want to establish an environmentally friendly protocol that is both specific and sensitive. The following two paragraphs outline the first developed direct tissue assay and our present assay. Former procedures: 30-50 mg of kidney tissue was collected and placed in RNAlater. A 20 mg piece of tissue was excised from the original sample and extracted with Trizol. The tissue was ground in a 1.5 ml microfuge tube using a nuclease free pellet pestle. Initially the Access RT-PCR System (Promega), then the Ready-to-go RT-PCR Beads system (Amersham and Promega) and ISAV 1D/2 primers were used for amplification. Present procedure: Tissue is collected as before and placed in RNAlater. Extraction is done using either the Sigma GenElute Mammalian Total RNA Miniprep kit or the Qiagen RNeasy kit. We decided against Trizol in part due to its toxicity to users and waste handling and in part due to the ease of use of the Sigma and Qiagen extraction kits. The tissue piece is homogenized with glass beads (710-1180 µm) in the supplied lysis buffer and RNA extracted following protocols indicated with the respective kits. Amplification is done using the EZ rTth RNA PCR kit (Perkin Elmer) and ISAV 1D/2 primers. This amplification kit worked equally as well as those listed above. It was chosen over the two former kits due to lower licensing fees. Our sensitivity with this procedure is presently at or below 0.1 pg using kidney tissue. SYBR Green is being used as an alternative to ethidium bromide for staining gels in order to reduce hazards to users and eliminate hazardous waste handling. Also, in addition to the ISAV 1D/2 primers, the FA-3 / RA-3 primer set is also used for RT-PCR in certain instances.

WORK PLANNED: Umaine: Sequence data is now available from the Anderson laboratory for ISAV genome segment 5, which encodes the nucleoprotein (NP). We will develop a RT-PCR assay for ISAV based on the sequences of genomic segment 5. This will result in the availability of five RT-PCR assays for the detection of ISAV: one assay each based on genome segments 5, 7, and 8 and two assays (including a nested reaction) based on genome segment 2. We will compare the sensitivity of all five RT-PCR assays for detection of ISAV in infected cell cultures as well as directly in fish tissues. Evaluation of the sensitivity and reliability of the RT-PCR assays for direct detection of ISAV in infected fish tissue samples will be conducted by a “blind” study in cooperation with MicroTechnologies, Inc. MicroTechnologies, Inc. will send us “blind” samples of both uninfected and infected fish tissue samples as determined by cell culture isolation. We will test each sample using all five RT-PCR assays and compare the results in regard to sensitivity and reliability with the conventional method of isolation in cell culture. Micro Technologies would like to shift funds for surveillance to research for optimizing the RT-PCR assay and field trial testing the optimal tissue submission for the detection of ISAV (other funds have been obtained for surveillance testing). Optimization strategies will focus on testing the efficiency and specificity of primer sets, along with examining amplification programs, annealing temperatures and salt concentrations. Field trials will examine both lethal and non-lethal samples to determine what organ/tissue is the most appropriate for screening for ISAV. Micro Technologies has initial data suggesting that blood samples are actually better than kidney samples when screening by RT-PCR and possibly culture. Lastly, should any funds remain after the optimization studies, we would like to begin validation studies for this diagnostic assay using protocols outlined in the Office of International Epizootics, Manual of Standards for Diagnostic Tests and Vaccines. IMPACTS: The results of this phase of the investigation will result in an optimal RT-PCR assay for the detection and identification of ISAV in fish tissue samples and determine the potential of the RT-PCR assay for use as a general diagnostic tool for the detection of this virus in aquaculture populations.


PUBLICATIONS, MANUSCRIPTS, PAPERS PRESENTED: Publications in Print: Anderson, ED, Engelking, MH, and Kurath, G.

2000. Molecular epidemiology of IHN virus within an Oregon study site reveals emergence of a new virulent virus strain and transmission from wild salmon to hatchery fish. J Aquat Anim Health 12:85-99.

Blake, S., J-Y. Ma, D. Caporale, S., S. Gupta and B.

L. Nicholson. Genomic variation and relationships among aquatic birnaviruses based on nucleotide and deduced amino acid sequences of cDNA of genome segment A. Diseases of Aquatic Organisms, in press.

Boettcher, K. J., B. J. Barber, and J. T. Singer. 2000.

Additional evidence that juvenile oyster disease is caused by a member of the Roseobacter group and colonization of nonaffected animals by Stappia stelulata-like strains. Appl. Environ. Microbiol. 66:3924–3930.

Chiou PP, CH Kim, P Ormonde, JC Leong. 2000.

Infectious hematopoietic necrosis virus matrix protein inhibits host-directed gene expression and induces morphological changes of apoptosis in cell cultures. J Virol. 74:7619-27.

Corbeil S, LaPatra SE, Anderson ED, Kurath G.

2000. Nanogram quantities of a DNA vaccine protect rainbow trout fry against heterologous strains of infectious hematopoietic necrosis virus. Vaccine. 18:2817-24.

Trobridge, G.D., C.H. Kim, S. Lapatra and J.C.

Leong. 2000. Mx mRNA expression and RFLP analysis of rainbow trout Oncorhynchus mykiss genetic crosses selected for susceptibility or resistance to IHNV. Dis Aquat Organ.40:1-7.

Johnson, M.C., B.E. Simon, C.H. Kim, and J.C.

Leong. 2000. Production of recombinant snakehead rhabdovirus (SHRV); the NV protein is not required for viral replication. J Virol. 74:2343-50.

Kim, C.H., M.J. Johnson, J.D. Drennen, B. Simon, E.

Thomann, and J.C. Leong. 2000. DNA vaccine encoding a viral glycoprotein induces non-

specific immunity and MX protein synthesis in fish. J. Virol. 74:7048-54.

Lapatra, SE., Corbeil, S., Jones, RG., Shewmaker,

WD., Lorenzen, N., Anderson, ED., and Kurath, G. 2001. Protection of rainbow trout against infectious hematopoietic necrosis virus four days after specific or semi-specific DNA vaccination. Vaccine in press

Leong, J. C., D. Brown, P. Dobos, F. S. B. Kibenge,

J. E. Ludert, H. Muller, E. Mundt, and B. Nicholson. 2000. Family Birnaviridae. In M. H. V. vanRegenmortel, C. M. Fauget, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, A. Mayo, D. J. McGeoch, C. R. Pringle, and R. B. Wickner (ed.) Virus Taxonomy: Seventh Report of the International Committee on the Taxonomy of Viruses, Academic Press, pp. 481-490.

Opitz, H.M., Bouchard, D., Anderson, E., Blake, S.,

Nicholson, B., and Keleher W. 2000 A comparison of methods for the detection of experimentally induced subclinical infectious salmon anaemia in Atlantic salmon. Bull. Eur. Ass. Fish Pathol. 20(1):12–22.

Opitz, H. M., D. Bouchard, E. Anderson, S. Blake, B.

Nicholson, and W. Keleher. 2000. A comparison of methods for the detection of experimentally induced subclinical infectious salmon anemia in Atlantic salmon. Bulletin of the European Association of Fish Pathologists, 20(1): 12-22.

Papers Presented: Nicholson, B. L. 2000. Applications of Molecular

Biological Tools in Diagnostics and Epidemiology of Viral Pathogens in Aquaculture, Jan. 13-14, San Diego, CA.

Nicholson, B. L. 2000. Applications of Molecular

Biological Tools in Diagnostics and Epidemiology of Viral Pathogens in Aquaculture, Sept. 28-Oct. 4, Townsville, Australia.




00-4 “Development of a Producer’s Practical Guide to Intensive Aquaculture” Progress Report Period: Febuary 1, 2001–June 15, 2001 NRAC Total Funding: $19,878 (August 1, 2000 – July 31, 2001) (No-Cost Extension through December 31, 2001) Principal Investigator: Michael B. Timmons, Cornell University Participating Investigators/ Cooperative Agencies:

Fred Wheaton University of Maryland Maryland James Ebeling Freshwater Institute West Virginia Steven T. Summerfelt Freshwater Institute West Virginia Brian J. Vinci Cornell University New York

PROJECT OBJECTIVES: The objective of this project is to develop a producer’s practical guide to intensive aquaculture (Producer’s Guide), which would provide a means to transfer practical engineering technology information from engineering researchers to aquacultural producers. Actions Requested:

1. none Progress: A rough draft of the book has been completed. We anticipate sending the book to the printers by October 31, 2001. The Cornell Freshwater Institute Summer Short Course was held. At this event, the Book as a Manual was used. A lot of good suggestions were given to improve the book and have been incorporated. Several publishers have been contacted. We expect the price of the book (approximately 700 pages) plus software will retail at $70.00. We are quite pleased about this.

Enclosure: I am attaching two chapters as word files for your review and providing the current table of contents:

Recirculating Aquaculture Systems: RAS Engineering

M.B. Timmons, J.M. Ebeling, F.W. Wheaton

S.T. Summerfelt, B.J. Vinci

Publisher: Cayuga Aqua Ventures, LLC. Ithaca, NY 14850

Table of Contents Foreword Chapter 1: INTRODUCTION

1.0 Background 1.1 Aquaculture Is Not for Everyone 1.2 Some Quick Case Histories 1.3 Other Experiences 1.4 The Optimistic View 1.5 Overview of Recirculating Aquaculture Systems 1.6 Water Requirements, Use, and Conservation 1.7 Market Dynamics 1.8 Interactions Between Objectives, Resources, Management, and Design 1.9 Terminologies and Nomenclature 1.10 Summary


Chapter 2: WATER QUALITY

2.0 Introduction 2.1 Water Quality Standards 2.2 Quantity 2.3 Sources 2.4 Parameters 2.5 Measurements

Chapter 3: FLUID MECHANICS, PUMPS, AND FLOW MEASUREMENT

3.0 Fluid Mechanics 3.1 Frictional Losses 3.2 Measurement 3.3 Pumping 3.4 Airlift Pumps

Chapter 4: LOADING and FISH GROWTH

4.1 Introduction 4.2 Production Terms 4.3 Water Quality Design Targets 4.4 Fish Growth 4.5 Design Examples

Chapter 5: CULTURE UNITS

5.1 Introduction 5.2 Stocking Density 5.3 Culture Tank Engineering 5.4 Round Tanks 5.4 Dual-Drain Structures for Concentrating Solids 5.5 Raceways 5.6 Carrying Capacity Issues 5.7 Stock Management Issues 5.8 Scale Issues 5.9 Mechanisms to Remove Dead Fish

Chapter 6: SOLIDS CAPTURE

6.0 Overview 6.1. Solids Balance 6.2. Basic Design Parameters for Round Tanks 6.3. Solids Generation 6.4. Total Suspended Solids (TSS) Physical Characteristics 6.5. Removal Mechanisms

Chapter 7: BIOFILTRATION

7.0 Introduction 7.1 Nitrification 7.3 Biofilters 7.4 Engineering Considerations 7.5 Basic Design Concepts – Fluidized-Bed Sand Biofilter 7.6 Basic Design Concepts – Floating Bead Biofilter 7.7 Basic Design Concepts – Trickling Tower Biofilter

Chapter 8: GAS TRANSFER

8.0 Introduction 8.1 Dissolved Gases –Basics 8.2 Gas Transfer 8.3 Gas Transfer Options

Chapter 9: SYSEM MONITORING AND CONTROL

9.0 Introduction 9.1 Parameters To Monitor 9.2 Monitoring Sensors And Equipment Options 9.3 Automatic Phone Dialers 9.5 System Design And Maintenance 9.6 Construction Hints

Chapter 10: WASTE MANAGEMENT

10.0 Introduction 10.1 Waste Management Issues 10.2 Waste Characteristics 10.3 Storage, Thickening, And Stabilization

Chapter 11: SYSTEM MANAGEMENT and OPERATION

11.0 Introduction 11.1 Backup Systems 11.2 Laboratory Facilities 11.3 Quarantine Facilities 11.4 Waste Management 11.5 Storage – Feed And Chemicals 11.6 Fish Product Handling 11.7 Transporting Live Fish


11.8 Purging And Off-Flavor 11.9 Post Harvest Handling 11.10 Feed Storage 11.11 Handling 11.12 Labor 11.13 Access 11.14 Operations 11.15 Miscellaneous Operations 11.16 Record Keeping And Maintenance

Chapter 12: STERILIZATION and DISINFECTION

12.0 Introduction 12.1 UV Irradiation 12.2 Ozonation 12.3 Factors Influencing Disinfection Efficiency 12.4 Characteristics Of Water Qualities

Subjected to Disinfection in the Salmon Aquaculture Industry

12.5 Inactivation of Fish Pathogens 12.6 Other Methods of Disinfection 12.7 Conclusions

Chapter 13: BIO-SECURITY AND FISH HEALTH MANAGEMENT PLAN

13.0 Introduction 13.1 Prevention 13.2 Biosecurity Protocols 13.3 Fish Health Management 13.5 Health Treatment Protocols 13.6 Responding To Disease Problems 13.7 Summary Of Methods To Ship 13.8 Treatment Of Diseased Fish 13.9 Calculation Of Disease Treatments 13.10 Fish Disease Diagnostic Services

Chapter 14: HEAT TRANSFER AND VENTILATION

14.0 Heat Transfer 14.1 Heat Transfer 14.3 Air Quality Control

Chapter 15: ECONOMICS AND RISK EVALUATION

15.0 Introduction 15.2 Case History of Fingerlakes Aquaculture LLC 15.3 Lessons Learned from Fingerlakes

15.4. Investment Choices 15.5 Species Selection 15.6 Scale Effects And Risk 15.7 Labor Requirements 15.8 Initial Costs And Economic Analysis 15.9 Comparison Of RAS Tilapia, Catfish Pond & Broiler Production

Chapter 16: FEEDS AND NUTRITION

16.0 Feed Management 16.1 Selection 16.2 Growth Performance 16.3 Feed Quality 16.4 Physical Characteristics 16.5 Practical Feed Formulation 16.6 Important Aspects of Aquaculture Feeds 16.7 Relationships Between Temperature, Body Size, Metabolic Rate and Feeding. 16.8 Relationships Between Water Chemistry and Dietary Needs 16.9 Functional Anatomy of Digestion 16.10 Minerals 16.11 Feedstuff Selection 16.12 Feedstuff Digestibility 16.13 Pelleted, Expanded, and Extruded Feeds

Chapter 17: SOFTWARE

• flow rate calculations based upon loading • pipe flow, orifice flow, pipe friction • Oxygen requirements • LHO design • CO2 control • sand filter design • cost analysis • feeding rate calculations and fish growth


REFERENCES AND BIBLIOGRAPHY APPENDIX

Industry Advisory Panel

Member Representation Affiliation John Ewart Extension University of Delaware Don Webster Extension University of Maryland Mike Iannello Industry Fingerlakes Aquaculture, Freeville, NY Jerry Redden Industry AquaMar, Pocomoke, MD Glenn Snapp Industry Water Management Technologies,

Baton Rouge, LA Terry McCarthy Industry Water Management Technologies,

Baton Rouge, LA Thomas Losordo Research North Carolina State University




00-5 “An Industry Directed Feasibility Study of the Razor Clam (Ensis directus) as a Candidate for Intertidal and Shallow Subtidal Culture in the Northeastern U.S.”

Progress Report Period: January 1, 2001–June 1, 2001 NRAC Total Funding: $76,900 (January 1, 2000 – December 31, 2002) Principal Investigator: William Burt, SouthEastern Massachusetts Aquaculture Center Participating Investigators/ Cooperative Agencies: William Burt SEMAC Massachusetts Dale F. Leavitt SEMAC Massachusetts Gregg Rivara Suffolk County. Mar. Env. Learning Ctr New York Gef Flimlin Ocean County Extension Center New Jersey Tessa Simlick Connecticut Sea Grant Program Connecticut Dave Alves RI-DEM Rhode Island James Rose Rose's Oyster Bar Massachusetts Michael Patricio Suffolk County. Mar. Env. Learning Ctr New York John Maxwell Atlantic City Aquaculture, Inc. New Jersey Les Hemmila Barnstable Sea Farm Massachusetts Carl Syriala Finn Shellfish Massachusetts Wentzle Ruml Wellfleet Sea Farms Massachusetts James O'Connell Wellfleet Massachusetts John Wadsworth Niantic Sea Farms, L.L.C. Connecticut Mark Zivan Nauset Sea Farms Masschusetts Richard Karney, Martha's Vineyard Shellfish Group Massachusetts Jeffrey Gardner Shellfish 4 U Rhode Island

PROJECT OBJECTIVES: The overall objective of this proposal is to provide an opportunity for the current shellfish culture industry to investigate, develop, and optimize the growout technology for a cultured razor clam (Ensis directus). This will be achieved through provision of small (one to two inch) seed razor clams, generated by a commercial hatchery, to participating growers and through distribution of sufficient funds to design, construct and test specific grow-out strategies, conceived and implemented by individuals currently practicing shellfish culture at a commercial scale. The specific objectives of the proposed work include: 1. Arrange with the participating commercial

hatchery to spawn and raise500,000 larval/juvenile razor clams (per year) to a one to two inch size for distribution within the project (Years 1 & 2),

2. Solicit ideas for technology to achieve razor clam grow-out from an array of commercial shellfish growers in Maine, Massachusetts, New York and New Jersey (Year 1),

3. Convene a committee of five individuals to select six shellfish growers (two from ME, two from MA, one from NY and one from NJ) for participation in the project, based on their concepts of razor clam growout technology (as solicited by the project) (Year 1),

4. Supply each of the six selected growers with 150,000 razor clam seed and $2,500 in operating funds (per year) to construct and implement their concept of appropriate razor clam growout technology (Years 1 & 2),

5. Design a data collection system that will permit comparable data to be collected at each of the growout sites, including biological and economic information, and instruct the industry participants on how to collect and record the required data (Year 1),


6. Task the industry participants to collect and relay the required data to the Principal Investigator for compilation and analysis (Years 1 & 2),

7. Identify sources and track economic data on razor clam markets within the region through the duration of the project (Years 1 & 2),

8. Compile the data for the term of the growout program (Years 1 & 2),

9. Analyze the data and project the overall benefits and limitations to the development of the razor clam as an alternate commercial aquaculture species in the northeast region of the US (Year 2),

10. Distribute the information throughout the region via the publication of a razor clam culture information bulletin, presentations and/or workshops directed at regional aquaculture associations and other industry venues, distribution of information through the NRAC Regional Extension Project, and presentations at academic symposia (Year 2).

ANTICIPATED BENEFITS: The outcome of this proposal is the development of technologies for culturing an alternate shellfish species and to make that technology available to shellfish growers. The industry currently relies on two species of shellfish to keep the industry moving. This places an inordinate amount of risk on the growers. By developing an array of shellfish species available for culture it will allow individual industry members to diversify their crop and/or change their crop to adapt to changes in the environment or the market. There are two "hidden" outcomes of this project. The first is we propose to jumpstart development of a commercial-scale hatchery procedure for razor clams. As is always the case, the shellfish growers can't grow a thing without a supply of shellfish seed to start their grow-out. By providing the funding to allow a commercial hatchery to generate juveniles of a new shellfish species while not interfering with their regular shellfish production schedule, this provides a significant benefit to the development of alternate shellfish species. The second hidden anticipated outcome of this project is associated with the lines of communication within the shellfish industry. The shellfish aquaculture industry relies to a large extent on "word of mouth" type of communication. The shellfish growers rely less on written materials and more on

discussion, observation, and demonstration. Through the connections associated with the NRAC Regional Extension Project and through the efforts of individuals such as Richard Kraus at ARC, the information generated by this project will effectively be distributed throughout the industry. As discussed above, there will be a technical bulletin generated as a result of this study and this bulletin will be available through written and electronic media, but the real value and the real deliverables are contained within the design of the project. By distributing the project across the NRAC region and by locating in within an existing industry member's facility, we are ensuring that the data generated by this project, how different techniques to grow razor clams may be successful or not, will be noted and discussed by the industry. If a grower is successful, you can be sure that the information will be distributed very rapidly across the grower's network via discussions with each other and via discussions between the grower and the regional extension agents. The project design has built into it a means to most effectively communicate these results. PROGRESS AND PRINCIPAL ACCOMPLISHMENTS: Objective 1: Arrange with the participating commercial hatchery to spawn and raise 500,000 larval/juvenile razor clams (per year) to a one to two inch size for distribution within the project. The Project Coordinator provided broodstock razor clams to the Aquaculture Research Corporation (a commercial shellfish hatchery in Dennis, MA) in January 2001. The hatchery successfully spawned four million razor clam larvae and raised them through metamorphosis to an early juvenile life stage. Upon delivery to the PC, there were approximately 150,000 - 20mm razor clam juveniles surviving. Objective 2: Solicit ideas for technology to achieve razor clam grow-out from an array of commercial shellfish growers in Maine, Massachusetts, New York and New Jersey. Presentations were made at the Northeast Aquaculture Conference and Exposition (Portland, ME) and the Milford Aquaculture Seminar (Milford, CT) to inform the shellfish farming industry about the upcoming project and to solicit design concepts for rearing razor clams. A Request for Proposals was distributed to shellfish growers in six northeastern states (ME, MA, RI, CT, NY & NJ) to solicit participants for the project. A copy of the RFP is included.


Objective 3: Convene a committee of five individuals to select six shellfish growers (two from ME, two from MA, one from NY and one from NJ) for participation in the project, based on their concepts of razor clam growout technology (as solicited by the project). The razor clam advisory committee, consisting of aquaculture extension agents from each of the six participating states, was convened to select participants from the pool of proposals received in response to the RFP. Nine shellfish growers were selected from five states. ME was excluded because there were no applicants from that state. Objective 4: Supply each of the six selected growers with 150,000 razor clam seed and $2,500 in operating funds (per year) to construct and implement their concept of appropriate razor clam growout technology, nine shellfish growers were selected and each received approximately 11,000 razor clam juveniles in August - September 2001. They also contracted to receive $3,500 over the two-year project to provide materials and supplies for their participation in the project. Objective 5: Design a data collection system that will permit comparable data to be collected at each of the growout sites, including biological and economic information, and instruct the industry participants on how to collect and record the required data, a draft data collection system has been designed and will be finalized and distributed to the participants prior to their first data collection event. Objective 6: Task the industry participants to collect and relay the required data to the Principal Investigator for compilation and analysis, to be completed when the data collection information is distributed. Objective 7: Identify sources and track economic data on razor clam markets within the region through the duration of the project, contact has been made with a commercial razor clam harvester who will provide us with the weekly market price of razor clams over the year and a list of local dealers who handle razor clams. Objective 8: Compile the data for the term of the growout program. To be completed.

Objective 9: Analyze the data and project the overall benefits and limitations to the development of the razor clam as an alternate commercial aquaculture species in the northeast region of the US, To be completed. Objective 10: Distribute the information throughout the region via the publication of a razor clam culture information bulletin, presentations and/or workshops directed at regional aquaculture associations and other industry venues, distribution of information through the NRAC Regional Extension Project, and presentations at academic symposia. To be completed. WORK PLANNED: The project will continue as outlined in the proposal. With the expanded list of participants, the razor clam distribution amounts will be decreased a small amount as will the total amount of funds allocated per grower for developing the culture technology. One addition is the participation of a second hatchery into the program. The new teaching shellfish hatchery at Mass. Maritime Academy will also participate in the razor clam spawning and early culture to provide back-up for producing enough juvenile razor clams for the project. IMPACTS: To be completed with the completion of the project PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED: Leavitt, D.F., W. Burt and D.C. Murphy. (2000).

The feasibility of commercially culturing the razor clam. (presentation at NACE, Portland, ME.)

Leavitt, D.F., W. Burt and D.C. Murphy. (In Press).

The feasibility of commercially culturing the razor clam. (Milford Abstract) J. Shellf. Res.

The Full Report with all the data, graphs and

tables is available at the NRAC office upon request.


VI. PROJECT SUPPORT INFORMATION


COMPLETED PROJECTS

97-2 “Expansion of the ‘Fishguts’ Fish Anatomy, Health and Necropsy Software Training Program for the Aquaculture Community”

OTHER SUPPORT YEAR

NRAC-USDA

FUNDING UNIVERSITY INDUSTRY OTHER FEDERAL OTHER TOTAL

TOTAL SUPPORT

1997/98 $52,129 $36,717 $88,846

1998/99 $19,362 $19,362

1999/01 $40,000* $40,000

TOTAL $71,491 $148,208 * Support from the Maryland Agricultural Experiment Station, University of Maryland College Park, College of Agriculture and Natural Resources. 97-7 “Developing a Sustainable Market for Northeast U.S.A. Aquacultured Products by Effecting Attitudinal Changes in the Foodservice/Restaurant Sector”

OTHER SUPPORT YEAR

NRAC-USDA


TOTAL SUPPORT

1997/98 $31,050 $31,050.00

1998/99 $15,100 $15,100.00

TOTAL $46,150.00 $46,150.00

98-4 “Determination of Optimal Swimbladder Inflation in Striped Bass Larvae Reared in Intensive Systems”

OTHER SUPPORT

YEAR NRAC-USDA


TOTAL SUPPORT

1997/98 $25,234 $8,800 $8,800 $34,034

1998/99 $30,819 $9,125 $4,964 $14,089 $44,908

1999/00 $28,589

TOTAL $84,642 $17,925 $4,964 $22,889 $78,942


98-6 “Development and Application of Multiplex PCR for Screening of Shellfish Pathogens”

OTHER SUPPORT YEAR

NRAC-USDA


TOTAL SUPPORT

1998/99 $64,719 $45,365 Supply Oysters

$45,365 $110,084

1999/01 $77,384 $47,635 $47,635 $125,016

TOTAL $142,103 $93,000 $93,000 $235,100

ONGOING PROJECTS 98-5 “Improving Intestinal and Renal Phosphate Absorption in Fish”

OTHER SUPPORT YEAR

NRAC-USDA


TOTAL SUPPORT

1998/99 $65,900 $26,520 $92,420.00

1999/01 $69,950 $27,850 $97,800.00

1999/00 $43,530

TOTAL $179,380.00 $190,220.00 98-7 “Aquaculture Curricula Resource Guide Publication and Distribution”

OTHER SUPPORT YEAR

NRAC-USDA

FUNDING UNIVERSITY INDUSTRY OTHER FEDERAL OTHER1 TOTAL

TOTAL SUPPORT

1998/99 $3,000 $14,924 $14,924 $17,924

1999/00 $1,744 $1,744 $1,744

2000/01 $7,000 $2,203 $2,203 $9,203

TOTAL $10,000 $18,871 $18,871 $28,871 1 Other support (to date) included the following: FY99: Massachusetts Department of Food and Agriculture, 208 hrs @$23/hr = $4,784; NEBHE, AQUA project, 416 hrs @$23.37/hr = $10,140. FY00: Massachusetts Department of Food and Agriculture, 25 hrs @$23/hr = $575; NEBHE, AQUA project, 50 hrs @ $23.37/hr - $1,168.50. FY01: Massachusetts Department of Food and Agriculture, 60 hrs @ $23/hr = $1,380.00; NEBHE, AQUA project, 15 hrs @ $54.84/hr = $822.60.


00-2 “Improving Larval Survival for Black Sea Bass Aquaculture”

OTHER SUPPORT YEAR

NRAC-USDA


TOTAL SUPPORT

1999/00 $67,855 $15,000

$15,000 $82,855

TOTAL $67,855 $15,000 $82,855 00-3 “Surveillance of Infectious Anemia Virus (ISAV) in the Northeast”

OTHER SUPPORT YEAR

NRAC-USDA


TOTAL SUPPORT

2000/01 $45,950

$60,000 $13,000* $73,000 $118,950

TOTAL $45,950 $73,000 $118,950 *Maine Agricultural and Forest Experiment Station 00-4 “Development of a Producer’s Practical Guide to Intensive Aquaculture”

OTHER SUPPORT YEAR

NRAC-USDA


TOTAL SUPPORT

2000/01 $19,878

$19,878

TOTAL $19,878 $19,878


00-5 “An Industry Directed Feasibility Study of the Razor Clam (Ensis directus) as a Candidate for Intertidal and Shallow Subtidal Culture in the Northeastern U.S.”

OTHER SUPPORT

YEAR NRAC-USDA


TOTAL SUPPORT

2000/01 $38,450

$6,000

$5,000 $11,000 $49,450

2001/02 $38,450

$2,000 $12,000

$5,000 $19,000 $57,450

TOTAL $76,900 $2,000 $18,000 $10,000 $30,000 $106,900

a p r 2001 september 1, 2000 through august 31,...

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