Progress Report, February 2003 A Proposal Submitted to NOAA/OAR’s National Marine Aquaculture Initiative ($300K Set-Aside)

Project Summary:

Development and Testing of a Recirculating Seawater Nursery System for the Culture of Bay Scallops, Argopecten irradians irradians for Ongrowing and Stock Enhancement or Restoration

National Marine Fisheries Service

Northeast Fisheries Science Center

Aquaculture and Enhancement Division

212 Rogers Ave.

Milford Laboratory

Milford CT 06460

Principal Investigators:

James Widman                                     Sheila Stiles, Ph.D.

(203) 882-6508                                      (203) 882-6524

james.widman@noaa.gov                       sheila.stiles@noaa.gov

Gary Wikfors, Ph.D.                                Ronald Goldberg

(203) 882-6525                                       (203) 882-6546

gary.wikfors@noaa.gov                           ronald.goldberg@noaa.gov

Barry Smith                                           Joseph Choromanski

(203) 882-6589                                       (203) 882-6579mith@noaa.gov                             joseph.choromanski@noaa.gov


            Duration:  2 years beginning ASAP

            Year 1 Funds Requested: $100,000

            Year 2 Funds Requested: $100,000

                        Total Requested:  $200,000

Note: Duration was modified so that Year 2 Funds could be split between Year 2 and Year 3

Techniques will be developed to culture large bay scallop seed in a recirculating seawater system with food supplied by a mass algal culture system.  Techniques for culturing finfish in recirculating systems are commercially viable, but are not currently utilized by the shellfish industry.

The economics of growing large bay scallop seed utilizing four different biofilters will be evaluated.  Water quality and algal additions will be monitored and adjusted by process control systems providing 24/7 coverage.  Each system (biofilter plus rearing tanks) will be evaluated for cost per scallop reared and the size at which it is most economical to rear them.  Survival and growth will be evaluated on a frequent periodic basis.

Grow-out strategies in the wild will be evaluated by deploying 25-mm, nursery-grown scallops in early spring, with expectations of producing 65-mm scallops by fall.  The feasibility of producing bay scallops with distinctive shell markings for field identification, through selective breeding, will be investigated and stock enhancement and restoration options for this species will be explored.


INTRODUCTION

Demand for seafood from population growth and increasing per capita consumption continues to increase annually, while the traditional capture fishery appears to have reached its maximum sustainable yield.  To satisfy the increasing demand for seafood, many analysts agree that aquaculture has the greatest potential for success.  Worldwide, aquaculture has also been used as a resource-management strategy to enhance standing stocks of finfish and shellfish whose populations have declined as a result of overfishing and/or habitat degradation.

A few marine aquaculture successes in the U.S. are salmon, oysters, and clams.  In the northeastern United States, the economic value of the commercial aquaculture industry exceeds $162 million per year and is increasing annually.  The largest components are oyster and Atlantic salmon production in the states of Connecticut and Maine, respectively.  We believe that farming of the bay scallop, Argopecten irradians irradians, has potential for success in our region as well because it shares two key characteristics with the other successful crops: rapid growth and high market value.  The native bay scallop adductor muscle, when available, sells for $8-19/lb. in the market, selling recently for $28/lb.  Further, supply from the wild fishery is very unstable.   Producing scallop seed in a land-based nursery system with a shell height of 25 mm and then on-growing from April - December in coastal waters has the potential to produce a six-gram adductor muscle.  This is approximately twice the size of the frozen Chinese product currently imported to the United States and would be of much better quality.  Demand at the wholesale seafood level exists for this product.

For aquaculture to help meet seafood demand in a manner consistent with NOAA stewardship responsibilities, scientific data must be combined with sound management practices in a coordinated program.  A recirculating seawater shellfish nursery that produces large seed could be the cornerstone of a commercial bay scallop aquaculture enterprise.  It also could be used to grow other bivalves such as oysters, clams, sea scallops, and mussels, and if the effluent is treated properly, non-native species as well.  The Milford Laboratory has constructed two major components of a recirculating seawater bivalve nursery: 1) an experimental algal mass production facility and 2) an experimental juvenile bivalve recirculating seawater nursery system.  Support is needed to examine the technical and economic feasibility of growing large-seed size scallops suitable for further grow-out either for enhancement or commercial production.  Although a previous attempt by Epifanio and Mootz (1976) to grow oysters to commercial size in recirculating systems failed, there have been significant advances made in the development of recirculating seawater systems.  We believe the advances made, and the fact that we are not attempting to grow scallops to market size in these systems, warrants further examination and evaluation.

The development of recirculating, land-based culture systems allows  environmentally-sound culture practices in biosecure systems.  Partial grow-out of animals takes place under controlled conditions on land and reduces the time the animals are in the wild  exposed to variable conditions.  Similarly, land-based marine aquaculture could reduce the risk of potential disease and genetic impacts upon wild stocks. Such systems require less water than normal flow-through systems, thereby reducing considerably the amount of effluent generated. The resultant waste-water effluent can be treated to kill any potential disease organisms, thus establishing a biosecure system. Although recirculating systems may be more costly to build and maintain, these greater costs may be more than offset by increases in productivity, lower financial risk afforded by greater control over production, and decreased susceptibility to predation and catastrophic losses due to storms, harmful algal blooms, and other adverse conditions.

One of the main impediments to commercialization of bay scallop aquaculture is the inability to obtain a profitable crop in one growing season, avoiding high levels of overwintering mortality.  Previous studies have shown that scallops can be reared in the wild to about 50 mm in size in one growing season (Rhodes and Widman, 1984).  An economic analysis of this strategy has shown that this size is marginal in terms of potential profit, chiefly because adductor muscle yield from scallops this size is modest - less than half the yield from a 70-mm scallop generally caught in the second year (Rhodes and Widman, 1984).  Our leading strategy for development of a bay scallop industry in New England is based upon the production of 65-mm scallops in one season without overwintering. This can be accomplished by field deployment of 25-mm seed scallops in early spring when water temperature rises above 15EC. This seed production can be accomplished during the winter in recirculating seawater systems.

In a cooperative project with the Marine Sciences and Technology Center of the University of Connecticut, and with their financial and technical support combined with in-house funds, we have constructed a greenhouse for large-scale culture of microalgal feeds - the Greenhouse for Research on Algal Mass Production Systems (GRAMPS).  Using solar energy, rather than artificial lights as in most shellfish seed farms, and physical stirring of cultures, cost of production for microalgal feed has been reduced from $300-400 per dry kilogram to about $35, or 10% of the cost reported by most hatchery operators.  Research needs for GRAMPS remaining include: 1) implementation and optimization of computer monitoring and control systems for main operating parameters,  2) improvement in long-term dependability of cultures, and  3) evaluation of water-quality issues affecting shellfish when large volumes of algal culture are pumped to densely-cultured shellfish. We also have assembled a recirculating seawater culture system for the grow-out of post-set bay scallops to about 25 mm in size.  The greenhouse and scallop nursery systems are linked together to provide algal foods to the scallops as required.  Both systems are operated by automated process- control systems.  Although recirculating systems are used in finfish aquaculture, these methods have not yet been transferred to shellfish aquaculture.  The next step in this phase of the project is to demonstrate the feasibility of  rearing scallops from post-set to  about 25 mm in size for potential use as seed in stock enhancement/restoration programs or for grow-out.  With the greenhouse in place, up to 5,000 gallons of algal feeds per day can be provided, using algal strains high in nutrition as determined by our phytoplankton research program. In the process of doing this, the economic costs of scallop production can be evaluated based on a bioeconomic model developed previously.

To produce shellfish seed economically, there is a need to recycle both the algae that have not been consumed on the first pass, and to minimize the energy expended in polishing the water (Widman, 1998).  Polishing the water may include activities such as filtration, heating or cooling, disinfection or detoxification.  Although growing shellfish in a recirculating system is possible, for it to be a commercial success it must also be economically feasible.  Research needs for our recirculating seawater shellfish nursery include:  1) determination of most efficient biofiltration system for shellfish culture,  2) maximum densities at which shellfish can be grown, and  3) size to which shellfish can be grown economically.

Aquaculturally-based stock enhancement presents an alternative strategy toward increasing bay scallop production.  Although coastal habitat degradation has reduced the number of available growing areas, many viable locations that can support growth, still remain.  Often, recruitment limitation is the obstacle for establishment of self-sustaining natural populations.  Using aquaculture to produce large numbers of seed scallops or spawner stocks can provide a way to overcome a lack of natural recruitment and eventually rebuild stocks.  Significant potential economic benefits exist for commercial fishermen with supplemental bay scallop harvest opportunities and for municipalities through the sale of recreational licenses.

a)  Current State of Knowledge

Although recirculating systems are used extensively in finfish culture, there is no commercial acceptance of recirculating systems for shellfish culture.  Finfish can be fed manufactured food, but shellfish require live phytoplankton as a source of nutrition. Although some advances have been made in producing algal pastes, they are expensive and still lacking in a complete diet.  Epifanio and Mootz (1976) grew oysters in a recirculating system but found it was not economically feasible to grow them to commercial size at that time.  Recent advances in technology and biofiltration warrant another look at the use of recirculating seawater systems for shellfish culture. Such systems would allow optimization of diets and growing environments to promote faster time to market, as has often been done by the agriculture industry. Advances in algal production techniques (Wikfors et al.,1998) and knowledge coupled with advances in how often to feed scallops (Wikfors, 2000) leads us to the logical next step of recirculating seawater culture of juvenile shellfish. Employing recirculating culture allows for better utilization of the algal biomass that the scallops require as food.

One of the most important aspects of recirculating systems is the selection of biofilters. A biofilter must convert waste products as well as allow algae to remain in the water column and available to the shellfish (Widman, 1999).  Pfeiffer (pers. comm.) experienced the problem of bead biofilters depleting the phytoplankton when culturing clams. We are aware that certain biofilters can filter and/or damage phytoplankton, and we will select filters which should minimize or eliminate these problems.  Although we hope to raise scallops to a shell height of 25 mm, our goal is to determine to what size juvenile scallops can be cultured economically in these recirculating systems.  By using biofilters that are commercially available or easily constructed, the technology will be readily transferable to commercial interests.

There is considerable demand in the market place for native-grown bay scallop adductor muscles. To date, culture of bay scallops depends on the production of hatchery seed that is immediately transferred to various types of flowing water systems at ambient temperature (Widman, 2001; Karney, 1994; Cunningham and Sun, 1995). When scallops are produced early in the season they may reach a size of 50 mm in the New England area before growth ceases during the winter months (Rhodes and Widman, 1984; Cunningham and Sun, 1995). Production of a 50-mm scallop in one season for the adductor muscle, however, is not economical at the present time.  Adams et al. (2000) concluded that a market for smaller whole bay scallops cooked and served in the shell could be developed.  Another impediment to commercial culture has been overwintering mortalities (Barber and Davis, 1997; Widman, unpubl. data). Bay scallop growth has been shown to be temperature-limited by Kirby-Smith & Barber (1974); to produce a larger scallop in a single season requires hatchery-produced seed, phytoplankton food ,and a temperature-controlled nursery. A bay scallop with a shell height of 65 mm should produce an adductor muscle twice the weight of a 50-mm scallop (Rhodes and Widman,1984). By producing a 25-mm seed scallop and deploying it in the natural environment by early May, so that it can take advantage of the full growing season, it should be possible to produce a 65-mm bay scallop by November/December.

There is much interest in the potential for farmed mollusks to “clean” coastal waters of suspended particles and nutrients through their filter-feeding activities.  While the only nutrients directly removed from the ecosystem are those contained within the tissues of harvested shellfish, scallops and other mollusks planted in coastal waters can have a profound affect upon the partitioning of nutrients, i.e., chemical form, particle size, and, thus, fate in the environment.  As nitrogen is the nutrient of greatest concern in coastal waters, this element has received the most attention.  Nitrogen contained in phytoplankton proteins removed from suspension by scallops can be digested or rejected in feces or pseudofeces, released as ammonia following catabolism, or incorporated into scallop tissue proteins.  As conversion efficiencies of scallops are in the range of 10-20%, most nitrogen consumed by a scallop will be returned to the environment in dissolved or particulate form.  Newell (pers. comm.) points out that particulate nitrogen in fecal and pseudofecal material, if deposited on oxic sediments, has the potential to be converted to dinitrogen gas by benthic microbial processes, thereby decreasing the quantity of biologically-available nitrogen in the ecosystem.  Partitioning of nitrogen by oysters is being investigated elsewhere (Newell, pers. comm.).  We propose to begin studies of nitrogen partitioning in bay scallops as part of this project.

In recent years, a general decline of available habitat and water quality, inimical algal blooms, and fishing pressure have likely contributed to a marked decline in bay scallop landings.  In many areas, a low-level of settlement and survival is observed annually, but large populations fail to establish, possibly indicating recruitment limitation.  Arnold et al. (1998) described the theoretical dynamics of persistent, but recruitment-limited  populations of bay scallops in Florida.  The relatively short 18- to 22-month life-span of bay scallops in the Northeastern U. S., in which adults usually spawn only one time (Belding, 1910), limits the potential for natural recovery, once a local population has declined.

Direct seeding of hatchery stock and establishment of spawner sanctuaries are two possible stock enhancement strategies for the bay scallop.  Direct seeding of shellfish, as an enhancement measure, often is appealing because of the simplicity of the concept.  Predation on directly-seeded scallops, primarily by crustaceans, is the greatest impediment to this approach.  Best survival is often observed when large scallops are seeded late in the year, when temperatures decline along with predation intensity (Tettelbach and Wenczel, 1993; Goldberg et al., 2000).  Results of these efforts, however, usually are highly variable and, in most cases, monitoring is insufficient to determine if there is a net gain in population size.  In one seeding effort on Long Island, New York, survival of planted hatchery stock was low at several sites (0-12%); however, 25% of the genotypically distinct scallop set of the next year was estimated to be the progeny of the transplants (Krause, 1992; Tettelbach and Wenczel, 1993).  This result provides support for the concept of directly seeding scallops that may provide “new” larvae into a natural system.

Spawner sanctuaries to enhance scallop populations were evaluated by Peterson and Summerson (1992), who found that recruitment was correlated positively to adult bay scallop abundance in North Carolina.  Similarly, a 258 % increase in scallop population size was demonstrated within one sound in North Carolina following transplantation of a spawner stock, compared to a statistically non-significant increase in a control area with no transplantation (Peterson et al., 1996).   An effective, suspended-cage spawner sanctuary, using plastic mesh  to protect scallops from predation, has been demonstrated in the Niantic River in Connecticut (Goldberg et al. 2000).  Knowledge of the local hydrography and the timing of natural spawning, that enables the prediction of larval settlement areas, are critical in selecting the locations of spawner sanctuaries.

One major obstacle to fully testing the effectiveness of stock enhancement for bay scallops is the inability to identify the progeny from a directly seeded or spawner sanctuary broodstock.  A possibility will always exist, if the population increases, that larvae came from another area, unless some morphological or genetic biomarker could be associated with the progeny.

Bay scallops have distinctive shell markings which vary in color from mostly dark gray or brown to white, with intermediate colors of orange and yellow.  Patterns can be striped, mottled or solid colors and in various combinations.  While use of these  shell markings or phenotypes has been suggested for identification of populations, definitive and systematic trials have been limited.   There is a paucity of data on such studies although Notata clams are an example of natural genetic markers being exploited in bivalves.  Kraeuter et al. (1984) conducted a study on shell color in bay scallops and found a genetic basis.  A follow-up study by Adamkewicz and Castagna (1988) determined that the genetic basis was expressed in two ways.  One was a dominant gene that determined the existence (or not) of a background color.  The other appeared to influence expression of an overlying color or pattern. More recent studies have suggested the use of genetic and  DNA technology to evaluate stock enhancement success  (Brown et al., 2000).  While there are  advantages to employing molecular genetics technology to investigate stock identification, procedures are more complex and results are not immediately apparent. Krause (1992) reported a contribution of 25% from seeded bay scallops to native populations in Peconic Bay, employing genetic and statistical methods.  A combination of allozyme analysis employing gel electrophoresis and statistical methods of discriminant and multivariate analyses was used in that project. Allozyme analysis by Xue (pers. comm.) verified genetic diversity and elucidated differences among several scallop populations.  In contrast, shell markings are readily visible and can be quantified easily with simple counts and reported by almost anyone based on observations in the field.  Furthermore, because there is a genetic basis, shell patterns would not be lost as applied or tethered tags and fluorescent dyes might be.  Bay scallops with distinct shell markings could be reared in a hatchery to increase the frequency of particular colors or patterns for easy identification.  The Milford Laboratory has successfully produced such scallops for small scale breeding projects and field trials (Stiles et al, 1998a; Choromanski et al., 1999).

b) Contributions of Study to Subject Area

Results from this study will increase our knowledge and ability to use recirculating systems for the culture of juvenile bay scallops.  This knowledge should be directly transferable to other shellfish species such as oysters, clams, sea scallops and mussels.  A comparison of biofilters will be made with an emphasis on their usefulness for shellfish culture. Our knowledge and ability to produce algae in mass production systems will be enhanced. An economic analysis of recirculating culture for bay scallops will be conducted. The results of the economic analysis will determine areas where further research and improvement will provide the greatest economic return. This study will also provide new insight into restoration and enhancement of species whose stocks have been depleted by overfishing and habitat destruction.

c) National Marine Aquaculture Initiatives Addressed

The following National Marine Aquaculture Initiative priority areas are addressed:  1) system engineering - design and operation of the recirculating juvenile shellfish nursery and algal mass production systems,  2) nutrition - (a). a pilot demonstration of the economics of mass production of algae and (b). growth and survival of juvenile scallops on cultured algae for a prolonged amount of time in recirculating culture,  3) product development - (a). demonstrate feasibility and increase reliability of low-cost mass algal production, (b). production of systems tailored to the culture of juvenile shellfish production and (c). can lead to a single-season adductor muscle market in the United States,  4) water re-use - using recirculating systems to minimize both intake water and effluent water, 5) genetics - demonstrate the utility of selectively-bred markers, and 6) marine species enhancement using scallops.

RESEARCH PLAN (YEAR 1)

a)  Objectives

The objectives of Year 1 are to advance significantly the culture techniques and promotion of commercially and recreationally important marine molluscan bivalve shellfish such as the bay scallop, identify and optimize practical operational parameters of recirculating, land-based culture systems for marine molluscan shellfish, and communicate findings to shellfish farmers.  The specific objective is to demonstrate the technical feasibility of using recirculating, land-based culture systems for the culture of juvenile bay scallops to a size for planting in the field for stock enhancement/restoration programs or for grow-out to market.

b.) Work Plan

Objective 1: To demonstrate the technical feasibility of using recirculating, land-based culture systems for the culture of juvenile bay scallops to a size for planting in the field for stock enhancement/restoration programs or for grow-out to market.

Hypothesis 1.1: A suitable algal mass culture system (GRAMPS), linked to the recirculating scallop nursery system, can be developed and evaluated.    

Proposed:

Algal strains from the Milford Microalgal Culture Collection, known from our previous research to be superior shellfish diets (Wikfors et al., 1996), will be mass cultured in the GRAMPS facility for feeding scallops in the nursery system.  Bacteria-free stock cultures will be amplified through indoor carboy culture and GRAMPS kalwall culture for inoculation into two 20,000-liter oval tanks in GRAMPS.  These large production tanks will be operated semi-continuously, with harvest and refilling alternating between the two tanks.  For production cultures, seawater from Milford Harbor will be passed through a tangential-flow capillary filter with sub-micrometer absolute removal of natural particles and fertilized with commercial “f/2" nutrient products.  Algal culture parameters, such as temperature and pH, will be monitored continuously by sensors wired to a PC.  Monitoring of algal cell density will be done by microscope counts of samples collected periodically; these same samples will be used to monitor for living contaminants, especially ciliates, that may affect culture performance.  Other Milford staff will monitor bacterial counts, specifically Vibrio’s, periodically.  Mass-balance calculations using nitrogen inputs (as nitrate in the nutrient enrichment) and nitrogen in algal-cell protein will be compared with dissolved-nitrogen autoanalyzer measurements to determine the nitrogen budget in the microalgal portion of the integrated system.  Periodically, production cultures will be analyzed for microalgal protein, carbohydrate, and total lipid, by wet chemistry, to evaluate nutritional quality.

Data collected by computer, as well as more conventional means, during scallop-rearing trials will permit us to evaluate GRAMPS culture performance under full-production conditions. Previous trials, at low and inconsistent usage rates, have identified a few potential problems, specifically related to nitrogen budget and living contaminants, that may or may not impact culture performance at full production.  Some experiments in smaller containers have, and will address, some aspects of these problems, but the need to evaluate effects of scale necessitate the trials identified in this proposal.  Lack of capability for replication at production scale (two 20,000-liter tanks) will limit data analysis to correlation and time-series statistics.  Testable hypotheses identified during full-production trials will be investigated experimentally at smaller scale, where practical.

Accomplished (Year 1 and continuing into Year 2):

Testing of the GRAMPS system –

Traditional hatchery methods of growing microalgae use batch cultures that last less than two weeks.  Many algal culturists are also satisfied with about 0.1x106 calls/ml algal density.  We have shown that algal cultures open to air can be grown far longer with a semi-continuous culture methodology.  Further, cell densities much greater have been shown repeatedly in the GRAMPS system.  Accordingly, improvements in culture dependability and productivity are possible and will be dependent upon development of effective management of living contaminants in the greenhouse (pilot-scale) environment necessary for commercialization of recirculating-seawater systems for nursery culture of bivalve mollusks.

The alga grown in the greenhouse during this period, unless otherwise noted, was Tetraselmis chui strain PLY429.  Cultures were started at 100 to 200 l in one of eight 500- l cylindrical tanks with inoculum from the Milford Laboratory Algal Culture Laboratory.  Cultures were increased in volume according to the cell growth cycle.  At least one of these 500-l cultures was then used to start a culture in 20,000-l production tanks.  These large cultures were, in turn, given additional filtered seawater and nutrients as the algal cells increased in number.  Eventually, most of the algal cultures were found to be contaminated with microscopic organisms that reduced the quality of the algal feed for the recirculating nursery system.  Research focused on methods to improve culture quality and duration.

Investigations to identify culture quality -- comparing several methods including visual interpretations of the appearance of a culture in situ:  It is desirable to have an index (poor, fair, good, very good, and excellent) of the culture, as seen macroscopically, that relates to the actual culture quality that is assessed microscopically and chemically.  Progress was made in relating such variables as algal cell number, bacterial load, contaminant population, and nutrient additions to gross culture observation using the above index.  This system can be used to streamline day-to-day culture assessments but retain a uniform and transferable ranking system.  Further, this system may serve to alert culturists to changes in culture “health” between routine sampling periods.

Summary of GRAMPS tank longevity and cell density --  Algal cell production of five large tank cultures were documented.  Longevity of these cultures before quality was determined to be unacceptable ranged from 45 days to 6 months.  One tank, with a 3-month useful life, had an average cell density of 1.4x106 cells/ml; whereas another, longer-lasting tank had an average cell density of 0.3x106.  Cell density likely is related to light input, which varies seasonally, thus, we are investigating relationships between season and productivity.

Growing Tahitian Isochrysis (T-ISO) in GRAMPS -- Isochrysis sp. has been found to be useful in the culturing of bivalve larvae. T-ISO was grown in a 500-l cylindrical tank.  It was started on 23 April 2002 and performed very well until it was found to be contaminated with a small blue green alga on 17 June 2002; tank life= 55 days.

Eight thermal probes to monitor culture, air, and ground temperature have been installed in GRAMPS. Programming has been completed to monitor all temperatures at once on a computer.  Further programming is needed to log these data for later reference.  The completion of the thermal probe system paves the way for the installation and operation of a pH control loop using CO2 to hold pH below a set point for optimum plant growth.  These two computerized data systems form the central portion of an automated control system for the greenhouse. Once complete, other components can be added with minimal additional programming.

Proposed Year 3:

Two periods of linked greenhouse-nursery operation will be supported in 2003.

Difficulty with contaminants:  Open algal cultures will usually be found, at some point in their lives, to have other organisms “contaminating” the algal culture.  Some of these invasive organisms will either out-compete or consume the desired algae in the culture while others coexist and do not reach any significant population.  When a contaminant is found in an algal culture there are several considerations to be made which can be used to influence culture strategy.  What is the contaminant?  Is it known to consume the algae or is it known to be benign? Benign contaminants include “monads”.  Contaminants that can be harmful to an algal culture, but are not always, include several types of ciliates including Euplodes sp.  Examples of contaminants that will out compete the cultured algae are some blue green algae.  Further, some of these blue green algae can be undetectable and then displace the desired algae within four days.

An ongoing analysis of the possible contaminant vectors, i.e., the way an undesirable contaminant can enter a culture and survive, is an ongoing endeavor.  So far, a single dominant mode of contamination has not been identified.  It is theorized that more than one mode of contamination exists, each with a very low probability of success.  Therefore, identifying the actual event of contamination and the success or failure of that invasive organism becomes very difficult.  This is further complicated when one considers the amount of contaminating inoculum into a culture that can be up to 20,000 liters volume.  Also, when can the contaminant be detected?  It could possibly be weeks or months before contaminants have a large enough population to be detected.  These investigations will be continued to their conclusion.  This represents a barrier to a truly reliable continuous supply of microalgae for whatever applications.

With the addition of a new Research Chemist to the staff, applied 100% to evaluation of nitrogen balance in algal and shellfish systems, research on water quality has begun in earnest.  A dysfunctional TRAX autoanalyzer has been restored to working order, and several experiments on nitrogen concentrations and speciation in algal cultures, nutrient sources, incoming seawater, etc. have been conducted with in-lab tanks.  In addition, we have developed flow-cytometric methods for counting algae, bacteria, and other particles in open-tank cultures; these methods produce results in seconds rather than days and permit much finer time-scale measurement of microbial community dynamics in intensive aquaculture systems than possible using conventional methods.  Several large experiments designed to determine nutrient and microbial dynamics in greenhouse tank cultures of phytoplankton will be conducted this year, with an eventual goal the establishment of hydraulic residence times that will maximize algal production and minimize dis-equilibrium conditions leading to poor water quality or contaminant problems often associated with “stagnant” tanks.

Hypothesis 1.2: There is a difference in waste removal efficiency by the different biofilters.

Hypothesis 1.3: There is a difference in survival/growth between systems.

Hypothesis 1.4: There is accumulation or degradation of algae due to the biofilters.

Proposed:

Scallops will be spawned and reared in conical tanks in our hatchery following methods described in Widman et al. (2001).  When scallops reach 1-5 mm in size, they will be transferred to downwellers in the recirculating nursery for on-growing in 10-Fm filtered seawater at 25EC, a temperature that promotes rapid growth.  We will test four different types of biofilters. Each of the four recirculating systems is composed of three trays (72" x 24" x 6"), one of the biofilter types, a deeper tank (48" x 24" x 30") to hold the downwellers, and numerous air lifts to move seawater throughout the system.  Actuated solenoid valves will deliver phytoplankton from GRAMPS based on real-time fluorometric measurements or at timed intervals using algorithms modified for this purpose.  By delivering the food through frequent small batches vs. pulse-feeding large amounts, we anticipate lower peak levels of nitrogenous waste products. Scallops will be reared in downwellers until they reach a suitable size for tray culture (10 mm).  While being grown in the downwellers and trays,  they will remain on the same biological filtration system.  Energy usage will be tracked on a daily basis by monitoring the amount of electricity and natural gas (heat) used in the recirculating system.  Scallops will be grown in four separate, but similar, systems utilizing different biological filters  to examine the effects of filter media and type on scallop production.  Algal levels will be monitored continuously by use of submersible fluorometers and calibrated to algal cell counts conducted two or three times per week.  Temperature, dissolved oxygen, conductivity, pH, ammonia, nitrite and nitrate levels will be monitored.  Growth and survival will be assessed on a weekly or biweekly basis.  Occasionally, 24-hour monitoring of nitrogen levels will also be conducted in order to determine if there is variation in these levels associated with light/dark cycles.

Accomplished: (Year 1)

Testing of recirculating shellfish nursery system -

Due to contaminant problems with the GRAMPS cultures and temperatures that would have been lethal (extremely warm weather on the east coast) to scallops we were unable to operate the recirculating system in Year 1.  Consequently, we decided to run some nutritional experiments in the laboratory which allowed us to maintain proper temperature control. We examined differences between GRAMPS and carboy grown Tetraselmis and its effect on bay scallop growth and survival (see Scallop Nutrition below).

Proposed Year 3:

Scallops and oysters will be reared in the nursery; Only three biofilters, RBC, Aquacube and Biocord will be tested along with one recirculating system without a biofilter to serve as a control.

Accomplished (Year 1 and continuing into Year 2):

Scallop Nutrition -

When GRAMPS was operating in the fall of 2000 we were able to examine GRAMPS vs. Milford carboy production algae and its effect on growth of scallops in the laboratory. The experiment started Oct. 30, 2000 and ended on Dec. 20, 2000. We used packed cell volumes to equilibrate the number of algal cells fed to each of six 15-l buckets each holding 30 scallops.  Growth differences were observed between the scallops fed the GRAMPS and carboy-produced algae (Fig 1).  Large aggregations of cells were observed in the GRAMPS-produced algae and it is not known how this affected the packed cell volumes. Further, it was unclear whether differences in scallop growth were attributable to nutritional differences between algal sources or differences in water quality associated with the different culturing methods.  These questions led to the design of the following experiment.

Figure 1.  Growth of scallops fed GRAMPS vs. carboy-produced algae in fall 2000.

Bar graph showing growth of scallops.

A multifactorial experiment was conducted to determine whether different methods of growing and/or the media used to produce Tetraselmis chui affected bay scallop growth. Eleven different treatments were examined: 1. Carboy algae cells in carboy media 2. Carboy algae cells in GRAMPS media 3. Carboy algae cells in sterile seawater 4. GRAMPS algae cells in carboy media 5. GRAMPS algae cells in GRAMPS media 6. GRAMPS algae cells in sterile seawater 7. No algae cells in carboy media 8. No algal cells in GRAMPS media 9. No algae cells in sterile seawater 10. GRAMPS algae as harvested 11. Carboy algae as harvested. To provide algae for treatments 1- 9 algae and media were separated and resuspended in the appropriate combinations. This was accomplished by centrifuging algae cultures, pouring off the supernatant, resulting in a very dense paste. Algal cells were mixed with the appropriate media or seawater to make up all 9 treatments. Media and seawater volumes were matched to the volumes of their counterpart in treatment 10 or 11.  Thirty scallops were haphazardly placed into each one of eleven buckets. Buckets were fed one of the rations described above.  Scallops were held at 25°C, a temperature that Kirby-Smith (1974) found maximized growth.  Each bucket was drained on a daily basis, new seawater and food were added to a total volume of 15 l.  On a weekly basis scallops were removed, measured for shell height to the nearest 0.1 mm and each bucket was cleaned. Initially, scallops were fed 1.56x10 9 cells and this increased to 1.6x10 9  cells as the scallops grew. The experiment started on January 22, 2002 and terminated 72 days later on April 4, 2002.

There was no significant difference in bay scallop growth for the first 22 days between groups fed Tetraselmis chui grown using GRAMPS and the traditional Milford carboy method. But by day 29, scallops fed Tetraselmis grown using the Milford carboy method were significantly larger (ANOVA P>.0321) than scallops grown using the GRAMPS production method. The superior growth of scallops fed the carboy grown Tetraselmis continued throughout the remainder of the experiment (Fig 2).  Tetraselmis  cells suspended in different media exhibited no significant effect on scallop growth. Minimal growth occurred in the unfed groups that had only media added to them, indicating there was some nutritional value in particles remaining in the media and seawater after autoclaving.

One unexpected result was the length of time the scallops survived in the unfed groups -- 29 days -- but by day 37 mortality started to increase (Fig 3). Survival decreased for the fed groups with time, possibly due to large numbers of algal cells being added at once, becoming bound with pseudofeces or settling and therefore being unavailable over the 24-hour feeding period.  There is also the possibility that a unialgal diet may be nutritionally deficient in some critical area.

Figure 2. Growth of bay scallops fed Tetraselmis produced via GRAMPS and carboy suspended in GRAMPs’,  carboy and seawater  media.

Bar graph 2 showing growth of scallops fed Tetraselmis.

Figure 3. Survival of bay scallops fed Tetraselmis produced via GRAMPS and carboy suspended in GRAMPs’,  carboy and seawater  media.

Proposed Year 3:

Experiments with commercially-purchased microalgal paste and live, cultured algae will be conducted to isolate effects of feed quality from those of nursery-system function.

RESEARCH PLAN (YEAR 2) Modified to Years 2 & 3 – (see cover letter)

a) Objectives 

Objectives of Year 2 are to: 1) continue to optimize the growth and survival of scallops in the recirculating nursery system, 2) (objective eliminated), 3) begin studies of nitrogen partitioning in bay scallops, 4) continue investigating the feasibility of producing , through selective breeding, bay scallops with distinctive phenotypes of shell markings for field identification, and 5) explore stock enhancement and restoration options for the bay scallop.

b) Work Plan

Objective 1: Continue to optimize the growth and survival of scallops in the recirculating nursery system developed in Year 1.

Hypothesis 1.1: There is a difference in waste removal efficiency by the different biofilters.

Hypothesis 1.2: There is a difference in survival/growth between systems.

Hypothesis 1.3: There is accumulation or degradation of algae due to the biofilters.

Accomplished:

Testing of recirculating shellfish nursery system -

Two tests of the recirculating system were made during 2002 with mixed success. One test was conducted using bay scallops and lasted 34 days. Three different biofilters, RBC (rotating biological contact), Aquacube and Biocord, were examined. The fourth biofilter, a fluidized bed, had to be removed due to its release of heat into the water causing elevated temperatures that were lethal to the scallops. Scallops were fed 2 l of algal cells from the GRAMPS system every half hour.   Scallops with an initial mean shell height of 1.0 mm were placed into downwellers in each of three recirculating systems. Initial stocking density was done by volume to counted volume ratio: each downweller was stocked at 100 ml. Scallops grew and survived well in each of the three recirculating systems.  After 21 days the scallops had increased to volumes that ranged from 132 to 176 ml (Table 1) with no noticeable mortality.  However, at our next sampling interval on day 34 we had suffered a catastrophic loss, 100% mortality, due to an absence of nutritious algae.  At day 30 the scallops appeared healthy although the algae appeared to be crashing at this time.

Up to this point there was little increase in nitrogen levels. Approximately 36 l of deionized water was added with a metering pump on a daily basis to minimize salinity changes.  Measurements of pH remained in the 7.6 - 8.0 range, with minor (0.1) inter day fluctuations, and no adjustments were made. During this trial one of the feeding valves in the Aquacube system failed and resulted in a decreased feeding level to the scallops in the Aquacube treatment. Valve failure probably is responsible for the decreased biovolume observed on April 24, 2002. The decrease in scallop biovolume noted for the Biocord system is probably due to mortality occurring earlier than that in the other two systems.

Table 1.  Total Biovolumes of Scallops in the Recirculating Systems

 

 

04/03/02

04/24/02

05/07/02

 

 Aquacube

100 ml

132 ml

154 ml

System

 RBC

100 ml

176 ml

145 ml

 

Biocord

100 ml

172 ml

100 ml

Juvenile oysters, Crassostrea virginica, were used in a second test due to warmer water temperatures which would have been lethal to bay scallops. In addition to the three biofilters used in the previous test we also utilized the fourth recirculating system that did not have a biofilter as a control. The oysters were placed into downwellers in each of the recirculating systems and were stocked on July 23 and allowed to acclimated for 1 week. Oysters had an initial mean shell height of 7.7 on August 6, 2002. Sampling consisted of periodically measuring oyster shell height to the nearest 0.1 mm and biovolume was measured.  On day 37 oyster mean shell heights ranged from 8.8 - 10.5 mm when a decrease in the nutritional value of the food was experienced.

Due to food limitations we were only able to run each test under growing conditions for 37 days or less. Unfortunately, that was not enough time to see any significant nitrogen loading in the recirculating seawater. Normally spikes in ammonia and nitrite levels are observed as the nitrifying bacteria become established in the biofilters. Low nitrogen levels observed may be due to two reasons: 1) There is a high probability of ammonia uptake by the living phytoplankton we were using as our food source. 2) At the time we expected to see an increase in ammonia levels we also experienced a catastrophic loss of our nutritious live food source which halted nitrogen production by the shellfish. At this time we are unable to make an informed conclusion on whether an ammonia peak similar to one found when operating finfish recirculating systems would be observed.

All of the recirculating systems exhibited similar growth and survival levels prior to experiencing feeding failures. For the 37- day period we observed no advantages when comparing one biofilter to another. During the Crassostrea virginica trial, one biofilter was removed from the system due to a mechanical failure. Even in the absence of a biofilter there was no noticeable increase in nitrogenous waste, although the test period was short.

Scallops and oysters grew well and survived in the recirculating systems. Although the duration of the experiments was shorter than we desired, the mechanical devices worked well. In conclusion, further testing of the system is required.

Proposed Year 3:

Work will continue on optimizing growth and survival of scallops in the recirculating nursery. Improvements also will be made to the system based on the results of running the bioeconomic model. Work will build upon the activities described for year one ( See Year 1 Work plan).  To minimize the impacts contaminants may have on the algal production and our ability to use it as a food source for the scallops we propose utilizing commercially available algal paste. Using algal paste should allow us to concentrate on the nitrogen cycle and efficiency of the filters while the greenhouse system is being examined.

Experiments with commercially-purchased microalgal paste and live, cultured algae will be conducted to isolate effects of feed quality from those of nursery-system function.

Accomplished:

Bacteriological testing of recirculating shellfish nursery system -

Bacteriological samples were taken from several  sources connected with the recirculating scallop nursery. Over 700  media plates were spread and counted from approximately 400 different samples. The primary focus of the monitoring effort  was to measure  the levels of  Vibrio spp. which may be found in food sources and ambient seawater supply lines. The level of these bacteria can be used as an indication of the general health of  bivalve larval cultures and  point to possible sources of pathogenic activity.

Results of the monitoring program include consistent high counts of Vibrio spp. for one week following periodic, hot, freshwater flushing of the seawater lines. These high bacterial loads may have resulted from the large amounts of dissolved and undissolved organic matter that remained in the piping system.  In addition, newly established mass algal cultures also would invariably show very high numbers of Vibrio spp (> 105 cfu/ml) soon after being started; however, the counts from these cultures would decrease to near 0 cfu/ml, usually after 4-8 weeks.

Proposed Year 3:

            Bacteriological testing will continue as appropriate.

Objective 2: Evaluate scallop grow-out strategies in the wild.

Hypothesis 2.1:  Scallops deployed at 25 mm in early spring will produce a 6-gram adductor muscle by late fall.

Hypothesis 2.2: Stocking density affects adductor muscle weight.

Hypothesis 2.3: Stocking density affects survival/growth.

Proposed Year 3:

We must eliminate Objective 2 from the workplan until problems with the greenhouse and nursery are solved and sufficient numbers of 25-mm scallops are available for early May outplanting.       

Objective 3Begin studies of nitrogen partitioning in bay scallops.

Hypothesis 3:   Bay scallops extract and modify nitrogen consumed in the form of microalgal protein.

Proposed:

Evaluation of nitrogen partitioning will be quantified in a bulk way by monitoring phytoplankton inputs and scallop outputs (ammonia, biodeposits, tissue growth) in the nursery system and constructing a nitrogen budget.  A “balanced” nitrogen budget will indicate that we have identified and quantified all important nitrogen sources and sinks in the microalgal-scallop trophic chain. Further, we will conduct focused studies, using individual scallops, to quantify nitrogen partitioning under experimentally-varied particle inputs, using both cultured microalgae and natural seston.  As a part of determining the overall nitrogen budget of the system, we will measure dissolved nitrogen in the effluent tanks containing known biovolumes of the seaweed, Ulva rigida ( i.e., prior to discharge into coastal waters). This ensures that the effluent from this system contains very little nitrogen (Goldberg et al.,1998). Additionally, we will measure the growth of the seaweed within the biofilter, and uptake of waste nitrogen will be determined by the heated biuret Folin method (Dorsey et al., 1977, 1978) as modified by Wikfors et al. (1984). A computer-controlled experimental molluscan-rearing system of original design (Smith and Wikfors, 1998) will facilitate focused experiments on scallop feeding. These studies will permit us to project likely environmental impacts (both positive and negative) of scallop grow-out in coastal waters

Accomplished:

Nitrogen Partitioning -

Semi-quantitative data were collected during some experiments, but the TRAX autoanalyzer was not functional for quantitative assessments of dissolved N species.

Proposed Year 3:

See Hypothesis 1.1 section.  Once N dynamics in algal systems are better understood, experiments will move on to quantifying N dynamics in the algae-mollusk food chain.

Objective 4: Investigate the feasibility of producing , through selective breeding, bay scallops with distinctive phenotypes of shell markings for field identification.

Hypothesis 4.1   Morphologically distinct markers in bay scallops can be produced, identified, and exploited  for large-scale tagging studies in stock enhancement efforts.

Hypothesis 4.2 Scallops will maintain morphologically distinct markers for the lifetime of the scallop.

Proposed:

Scallops with distinctly visible (physical) markers such as striped or orange shells will be used to investigate  the reproductive success or genetic contribution of transplanted populations to stock enhancement efforts.   Scallops selectively bred for these distinctive phenotypic markers will be used in Objective 5 to field-test the feasibility of using these markers in assessing the success of stock enhancement efforts  (Stiles et al., 1998b; Choromanski et al., 1999).  This breeding approach will entail collecting, conditioning and spawning adults, culturing larvae and maintaining juveniles up to the adult stage in our Milford facility (Widman et al., 2001).  The selective breeding strategy will include spawning scallops which exhibit distinctive shell markings, primarily striped shells (since some bright shell colors may be more susceptible to predation), to increase the frequency of these phenotypes, which, in turn, are relatively low in nature.  Estimates of increased frequencies will confirm the success of stock enhancement efforts.  Scallops will be field tested and monitored for survival, growth, and retention of the shell color and markings.

Accomplished:

Selective breeding -

Selective breeding studies were initiated employing scallops with striped shells as markers for stock enhancement, as one major limitation to previously conducted stock enhancement  programs has been a lack of  identification of stocks.  The objective of this project is to investigate the feasibility of producing, through selective breeding, increased numbers of bay scallops with distinctive phenotypes for field identification.  These naturally-occurring scallops with distinctively visible markers  at low frequencies of  1 - 5% are being developed to determine the reproductive success or genetic contributions of transplanted populations to stock enhancement efforts possibly in sanctuaries. 

Broodstock scallops were brought into the laboratory and were conditioned for spawning.  Scallops with striped shells were selected to produce progeny with striped shells as phenotypic markers for stock identification in the field (Fig. 4).  The first spawn to develop scallop lines with striped shells was initiated in March 2002.   A second spawning was conducted in April 2002.  Striped shells were evident in scallops at 1 mm. in shell height.  The purpose of the initial phase was to evaluate these selected scallops for growth and survival.   Preliminary results indicate a positive response to selective breeding with an increased frequency of at least 50% of scallops with striped shells, and favorable growth and survival.

Fig. 4 – Selectively bred striped scallops

Photo of stiped scallops

A field trial was conducted to evaluate: 1) Scallops with and without striped shells for growth and survival and 2) Overall performance (growth and survival) of scallops in cages at two proximate, but dynamically different sites in the Niantic River of eastern Long Island Sound (Fig. 5). The 2 selected sites represent shallow and deeper water habitats.

Fig. 5 – Chart of the Niantic River estuary

Color map of Niantic River estuary.

Fig. 6 –Cages used in field experiments

Color photo of three tiered cage.

The laboratory-spawned bay scallops were evaluated in commercial, rigid-mesh cages.  The cages were made of plastic-coated wire with a 7.5 cm mesh (Fig. 6). Each cage measured 56 x 56 x 94 cm and was divided horizontally into three sections or tiers. Two ballast areas below the bottom tier provided an offset from the sea floor of approximately 15 cm. Cage inserts of smaller mesh (10 and 17.5 mm) measuring 41 x 10 x 81cm were used to hold the scallops.

The scallops were held in temperature-controlled tanks in the Milford Laboratory and in outdoor raceway tanks, using densities determined as optimal.  Subsequently, scallops from a selected line were measured and divided among the 3 shelves or tiers in 3 cages at each of the 2 sites.  One-hundred scallops, 50 with stripes and 50 without, approximately 30 mm in height, were put in each shelf of the cages making a total of 3 X 6 = 18 X 100 = 1800 scallops.  Cages with scallops were placed at the sites in late September, and retrieved 2 months later prior to initiating the overwintering phase.  Results from the field experiment indicated that satisfactory growth of scallops did occur at both sites in the Niantic River (Table 2).

Table 2.  Comparison of growth and survival of scallops with striped and non-striped shells at two sites in the Niantic River. (shell height -  mm; n=count)

Site 1          Striped                                         Non-Striped

 Tier                            (n)                                                    (n)

  A           42.87 + 0.76 (37)                             40.52 + 1.09 (37)

  B           38.31 + 1.36 (46)                             37.09 + 1.41 (39)

  C          33.94 + 1.30 (40)                             37.04 + 1.31 (33)

Site 2          Striped                                         Non-Striped

 tier

 D           40.28 + 0.82 (37)                             40.81 + 0.84 (28)

 E            38.43 + 1.17 (42)                             40.45 + 0.90 (24)

F            40.09 + 1.17 (44)                             35.38 + 1.36 (35)

 

Proposed Year 3:

We will continue evaluation of scallops with distinctly visible (physical) markers such as striped or orange shells to investigate the reproductive success or genetic contribution of transplanted populations to stock enhancement efforts (Stiles et al 1998a; Choromanski, et al., 1999).   Scallops selectively bred for these distinctive phenotypic markers will be used (in Objective 5) to field-test the feasibility of using these markers in assessing the success of stock enhancement efforts   (Stiles et al. 1998a; Choromanski, et al., 1999, Widman et al., 2001). This breeding approach would entail collecting, conditioning and spawning adults, culturing larvae and maintaining juveniles up to the adult stage.   The selective breeding strategy will include spawning scallops  which exhibit distinctive shell markings, primarily striped shells (since some bright shell colors  may be more susceptible to predation), to increase the frequency of these phenotypes.   The frequency of these phenotypes is relatively low in nature.  Estimates of increased frequencies will confirm the occurrence of these transplants based on recovery rates.  Scallops will be field tested and monitored by free planting as well as in cages, with an initial size (about 25-40 mm) that is suitable for probable survival and growth in the field.   Results will be analyzed.   Preliminary genetic investigations of bay scallops by breeding, allozyme, molecular and morphological analyses already indicate potential success for stock identification (Stiles et al., 1998b, Xue et al., 1995).

Field Density Experiments Late Juvenile to Adult Scallops

Scallops at approximately 25 mm will be counted and measured in preparation for field experiments in cages at the 2 sites in the Niantic River.  The number of scallops will be 100 per shelf X 3 shelves.  Scallops from genetics lines were also available for an overwintering experiment which began in November 2002.  Other scallops will be maintained concurrently in the tank farm at an optimal density.   The experimental design and statistical analysis will be ANOVA.  Scallops will be measured and otherwise evaluated for retention of genetic markers for the duration of the experiment.  Environmental evaluations (i.e., temperature, salinity, D.O., food availability) also are planned.

Objective 5: Explore stock enhancement and restoration options for the bay scallop.

Hypothesis 5.1:  Morphologically distinct hatchery-reared scallops (>40 mm) directly seeded into selected habitats in late fall or early spring will survive, grow, and eventually spawn.

Hypothesis 5.2:  Morphologically distinct hatchery-reared scallops protected from predators in a suspended-cage spawner sanctuary will grow and spawn.

Hypothesis 5.3:  The fate of the larvae introduced through direct seeding and spawner sanctuary experiments that recruit into natural habitats can be determined.    

Proposed:

Survival, growth, and reproductive state will be determined for morphologically distinct hatchery-reared scallops directly seeded within two different estuaries in three similar (replicate) habitats at different locations.  Simultaneously, these parameters will be measured in scallops maintained in suspended cages on long-lines, deployed within the same two estuaries in 3 habitats. Estuaries selected will be Niantic Bay and coastal Stonington in Connecticut, which have natural habitats where scallops have been harvested historically, but are not abundant presently. Survival of directly-seeded scallops will be based on diver counts along at least 10 replicate 25-m long x 2-m wide (field of view) transects.  All deployments will use large scallops (> 40 mm) and will be conducted in late fall or early spring, when predation pressure is presumably reduced.   These sites will be located in small embayments with limited tidal currents, where retention of larvae during the 7-9 day free-swimming period before settlement would be predicted (Kollmeyer, 1972).  Gametogenic development will be measured by visual inspection of developing gonads of at least 30 individuals from each treatment and scored based on the distinct 6-stage descriptions of Sastry (1963) and expressed as percentage of the scallops at each stage.  Prior to the time when spawning is likely, two replicate mesh-bag spat-collectors will be deployed in four areas surrounding the planted scallops and the long-lines.  Spat collectors will be inspected biweekly and replaced with new bags.  Presence of distinctly marked and unmarked spat will be counted.  At several intervals (i.e., prior to, during, and after spawning), statistical comparisons of survival, growth, and percent gametogenic development will be conducted, using an ANOVA block design (i.e., 2 estuaries  X  3 replicate habitats  X  2 treatments - free-plant and suspended cage).   Spatfall also will be compared for different collector deployments using ANOVA.   Data are likely to be arcsine transformed to achieve normality and meet assumptions of ANOVA.  Habitat quality, structure (eelgrass density), environmental conditions (DO, temperature, salinity), and predation levels will be characterized.  As in previous projects, we will seek cooperation from local Municipal Shellfish Commissions (Waterford - East Lyme and Stonington) to maintain and monitor experiments and for their valuable insights into local conditions.  The fate of the seeded, morphologically-distinct scallops, and possibly their progeny with inherited traits, will be followed in subsequent years through catch surveys of license holders.

Accomplished:

Scallop enhancement -

In 2002, new laws were implemented by the state of Connecticut that require any fixed aquaculture gear to be deployed in state waters be approved and permitted. The Lab=s research projects are not exempt, including the bay scallop stock enhancement project in the Niantic River.  Application for an Aquaculture Permit was submitted and reviewed by: State of CT Dept of Agriculture- Bureau of Aquaculture, DEP Headquarters in Hartford, DEP Fisheries, Boating Safety, as well as the US Army Corp of Engineers, Fish and Wildlife Service, and NMFS Habitat Protection Office. In addition, a presentation was made to two local shellfish commissions, who after consideration, wrote letters of endorsement.  The process took about 8 months and the permit was granted in September of 2002.

A population of scallops was produced through selective breeding (Objective 4) with a high frequency of stripes, a visible trait, useful in potential stock enhancement efforts.  About 3000 of these morphologically distinct striped bay scallops ( >40mm) were deployed in September 2002 and are currently being overwintered in the Niantic River (in cages) and in the laboratory.  The two sites chosen in the Niantic River (Fig 5) are areas where icing is generally not severe.  Survival will be assessed in early spring.

Proposed Year 3:           

April 2003 

A field experiment will be conducted to measure growth, survival, gametogenic activity, spawning, and settlement of caged and free-planted bay scallops, using those scallops produced in Objective 4, currently being over-wintered in the Niantic River.  At each of the 2 sites, we have established, 2 treatments will be maintained, a cage spawner sanctuary and a free planting. 

At each of the 2 sites, about 900 caged scallops will be deployed (divided among 3 cages with 3 tiers at 100/tier).  A free plant of about 300 scallops, adjacent to the cages, will also be established. 

Habitats will be defined spatially by recorded GPS coordinates.  A data logger will be maintained near the experimental sites to monitor temperature, salinity, and dissolved oxygen throughout the experiment.  Additionally measurements will be taken on site visits with a hand- held unit.

An additional field treatment of scallops confined within a fenced corral area will be implemented to gain insight into predation and movement of free-planted scallops.                                                                                                                

April - August 2003

Growth and survival will be measured in each treatment by sub-sampling caged and free planted scallops.  Survival of free planted scallops will be determined by SCUBA diver counts along transects.  Gametogenesis and spawning will be determined by visual observation of scallops and scoring based on the scale proposed by Sastry. 

Site characterization of physical habitat, vegetation, assessment of epihytic growth on vegetation, and crustacean predators will be based on visual SCUBA observations.  Generalized measurements of predominant current speed and direction will be made with a Marsh-McBirney meter.  These and environmental data will be incorporated within a GIS-based site map.

As gametogenesis progresses an attempt may be made to conduct a Afield spawning@. This would enable control of the timing of larval release.

July - September 2003

Spat bags will be deployed in the vicinity of each of the 2 spawner sanctuaries and monitored periodically for the presence of recently settled bay scallops. Replicate pairs of collectors will be deployed in a 4 x 4 array, which may allow empirical determination of larval distribution.  Spat will be quantified and possibly grown-out at the laboratory to determine if morphologically distinct traits are represented. Additionally, diver surveys will detect any spatfall on naturally occurring eelgrass.

c) Role of Project Personnel

This project will be conducted primarily with in-house staff, including culturists, disease specialists, algal nutritionists, geneticists, and ecologists.In year three, a newly hired Research Chemist, Dr. Shannon Meseck, will be working on the project as well.

OUTPUT 

Results of this research will further our knowledge of recirculating seawater systems for the culture of shellfish, with a particular focus on bay scallops.  It also will assist in the development of a commercial bay scallop aquaculture industry in the Northeast region, and other  shellfish nurseries, most notably clam and oyster, would directly benefit, since they could begin applying the results immediately.  Having the ability to rear shellfish in recirculating systems would allow researchers to perform genetic manipulation experiments while containing the experimental shellfish in a closed and biosecure system. Producing larger seed for enhancement by demonstrating successful stock enhancement, including the use of morphologically distinct scallops, will enable local communities to conduct long-term rebuilding projects aimed at rebuilding self-sustaining populations.

Results of the research will be presented at the Annual Milford Aquaculture Seminar, organized by the NMFS Milford Laboratory in February 2002 and 2003.  This seminar is held annually to provide technology transfer to the aquaculture industry.  Periodic progress reports and significant findings will be posted on the Milford Laboratory and DOC/NOAA Aquaculture Information Center website.  Results also will be presented at national and international aquaculture meetings and published in the peer-reviewed literature.  Advice and training will be provided to industry through personal contacts and hands-on training at the Milford Laboratory.  In cooperation with the Connecticut Sea Grant College Program, we will publish a leaflet on scallop grow-out and enhancement strategies.

Project Accomplishments

1)      Demonstrated biological feasibility of rearing bay scallops and eastern oysters in a recirculating seawater nursery on cultured microalgae

2)      Demonstrated that the nutritional value of greenhouse-cultured Tetraselmis is approximately 75% that of carboy-cultured algae, with greenhouse algae costing only10% of carboy algae to produce.

3)      Initiated studies of nitrogen speciation and mass balance in microalgal tank cultures feeding the scallop nursery; this effort is led by a newly-hired Research Chemist.

4)      Documented seasonal variance in production and dependability of greenhouse microalgal feed cultures; identified living contaminants management as most pressing research priority to improve the microalgal feed production process.

5)      Successfully employed selective  breeding and increased the frequency of genetic markers or striped shells in scallops  from 1 - 5% occurring  naturally in a population to 50% in the selected population. 

6)      Demonstrated that overall, scallops with and without striped shells exhibited good survival and growth, based on preliminary results.

      7)   Demonstrated that in general the 2 sites in the Niantic River were comparable for the performance (growth and survival) of scallops.

Publications and Presentations:

Widman, J.C. Jr., D. Veilleux  2001 Growth and Survival of Bay Scallops,
Argopecten irradians irradians, fed Tetraselmis chui Grown by Two Methods.
Journal of Shellfish Research, vol. 20, no. 1, p. 529.
Presentation for 23rd Milford Aquaculture Seminar February 2003.
Widman, J.C. Jr., D. Veilleux  Demand Feeding of Bay Scallops, Argopecten
irradians irradians Using an Automated Control System. 

Presentation for 23rd Milford Aquaculture Seminar February 2003.
Stiles, S.,J Choromanski,and D.Jeffress."Genetic Strategies for Culture and Stock Enhancement of Bivalves." 

LITERATURE CITED

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Arnold, W.S., D.C. Marelli, C.P. Bray, and M.M..Harrison. 1998. Recruitment of bay scallops Argopecten irradians in Floridian Gulf of Mexico waters: scales of coherence. Mar. Ecol. Prog. Ser. 170, 143-157.

Barber, B.J. and C.V. Davis.1997. Growth and mortality of cultured bay scallops in the Damariscotta River, Maine (USA). Aquacul. Int. 5:451-460.

Belding, D. L. 1910. A report on the scallop fishery of Massachusetts, including the habits, life history of Pecten irradians, its rate of growth and other facts of economic value.  Special Report, Commissioners on Fisheries and Game, Commonwealth of Massachusetts Publication.  Wright and Potter Printing Co.  Boston 150 p.

Brown, M.V., L. Strausbaugh, and S. Stiles.  2000.  Methodology for the generation of molecular tags in Placopecten magellanicus (sea scallop) and Argopecten irradians (bay scallop).  J. Shell. Res.  19: 569.

Choromanski, J, S. Stiles, C. Cooper, E. Beden., S. Lonergan and P. Trupp.  1999.  Growth and survival of  juvenile bay scallops from genetic lines at different densities and depths: Collaborative study between the NMFS and Bridgeport Aquaculture School.  J. Shell. Res. 18: 263.

Cunningham, T. and L. Sun.1995. Demonstration long-line scallop farm. J. Shell Res. 14: 240-241.

Dorsey, T.E., P.W. McDonald, and O.A Roels. 1977. A heated-biuret Folin protein assay which gives equal absorbance with different proteins. Anal. Biochem. 78:156-164.

Dorsey, T.E., P.W. McDonald, and O.A. Roels. 1978. Measurements of phytoplankton-protein content with the heated-biuret Folin assay. J. Phycol. 14:167-171.

Epifanio, C. E.and C. A. Mootz.  1976.  Growth of oysters in a recirculating maricultural system. Proc. Natl Shell. Assoc.,  65: 32-37      .

Goldberg, R., P. Clark, G.H. Wikfors, and M. Shpigel. 1998. Performance of Ulva rigida as a biofilter in a flow-through mariculture system. J. Shell. Res. 17:354-355.

Goldberg, R; J. Pereira,and P. Clark..2000.  Strategies for enhancement of natural bay scallop,             Argopecten irradians irradians, populations; A case study in the Niantic River estuary, Connecticut, USA. Aquacul. Int., 8: (2/3) 139-158.

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Budget Justification Year 3

Permanent Equipment
Laptop computer 2.0 K
Used to collect data and program data logger in the field

Digital underwater video camera 5.2 K
For quantifying scallop survival and density in the field.

Supplies

Algal research 7.0 K
Items required for algal production and nutrient analysis
including nutrients, filters, reagents, bacteriological, and
miscellaneous supplies.

Scallop research
Components for operation and upgrade
of recirculating seawater system, including DI water
recharging, larval rearing, peristaltic pumps, actuated
valves, blowers, and algal paste. 15.8 K

Genetic research
Digital thermometers, calipers, air pumps, pneumatic
control valve, dissecting microscope and light source,
photographic processing 6.7 K

Field enhancement research
Additional cages, nets, line, floats, blocks and hardware
for field deployments, wire mesh material for corral,
repair of data logger, batteries, membranes, 4.0 K

Hazardous Duty (20 dives, 2 divers) 2.0 K

Meeting Travel (for 4 individuals) 6.0 K

Publications 1.0 K

TOTAL 49.7 K

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