A. Brief Project Overview:
The Science Consortium For Ocean Replenishment And Enhancement (SCORE) is a science-based approach to stocking hatchery-reared marine organisms to help rebuild depleted marine fisheries (marine fisheries enhancement). SCORE scientists are conducting research to resolve critical uncertainties about the effectiveness of culture-based marine enhancement as a fishery management tool. It is anticipated that significant progress will be made in the next five years, leading to greater and greater success from marine enhancement programs in the U.S.

As scientific gains are made in understanding the potential, SCORE scientists are partnering with NMFS and regional fishery-management agencies to develop policy and apply fishery-enhancement science to rebuilding depleted coastal stocks. Linkages with local fishing communities provide the cadre of citizens needed to support and expand enhancement as a fishery management strategy. Much of the enhancement technology developed here will be supported by funds generated by contributions and license fees paid by stakeholders and user groups. To fully embrace and use the marine enhancement concept, demonstrated success stories are needed in a few key states. SCORE research is planned and coordinated to achieve such successes. Built around the principles of a responsible approach to marine stock enhancement (Blankenship and Leber; and see Leber, 2002), SCORE scientists are conducting key experiments to resolve critical uncertainty about how to control the biological, ecological, and economic effectiveness of marine fisheries enhancement.

SCORE is an R&D initiative conducted by a consortium of national partners. It is a powerful alliance of scientists and fishery managers currently working in the field of marine stock enhancement in the U.S.A., which encourages improved utilization of their expertise and resources. Bringing these scientists and managers together through SCORE allows synergisms to develop that would not occur otherwise.

This interim report covers progress made by SCORE during the period July 1, 2003 through December 31, 2003.

B. Project Accomplishments:

a. Tasks scheduled for this period
i. Develop aquaculture production technology
ii. Develop optimal stocking strategies
iii. Evaluate the effectiveness of stock enhancement
iv. Develop habitat/release model for winter flounder
v. Develop adaptive management strategies
vi. Identify released hatchery fish
vii. Describe life history patterns and ecological interactions

b. Tasks accomplished for this period
i. Develop aquaculture production technology
Development of a year round captive spawning protocol for common snook
In early 2003, a light and temperature controlled broodstock holding facility was constructed at Mote Marine Laboratory’s main campus. Two broodstock tanks (17,385 liter) were set up to hold snook broodstock. Each broodstock holding system was equipped with a bead filter for solids filtration, a fluidized bed for biofiltration, a UV unit, and a heater/chiller to control temperature. All snook broodstock at MML were pit-tagged to track growth and sex distribution. In 2003, we also constructed three new snook broodstock holding systems (54,315 L/tank) at Mote’s new aquaculture facility Mote Aquaculture Park (MAP). The construction and design of these systems was supported with funding from Mote Scientific Foundation and the Selby Foundation. The design for these systems took over a year to complete because these large-scale broodstock maturation and spawning tanks are located 17 miles away from the coast and will be operated on a closed-cycle or 100% recirculation. We plan to stock the first tank in February 2004 with broodstock from Mote’s interim broodstock holding facility on the laboratory’s main campus. Once stocked, attempts to induce spawning using temperature and photoperiod manipulation will begin.

Determination of an optimal diet for common snook broodstock
Approximately 26 captive broodstock fish were collected from February to May 2003 and are being held at MML’s main campus. These fish have been weaned off of live prey organisms and are now being fed a fresh cut broodstock diet of shrimp, kapelin, squid, herring, and mackerel. These fish are monitored for egg development and growth every quarter.

Reduction of cannibalism during rearing of common snook and the development of a cost-effective larval rearing culture technology for common snook
In 2003, three recirculating experimental systems were constructed that consisted of four cone-bottom cylinder tanks (114 L/tank). Each system had its own sump (that acted as a settling chamber for solid waste), a UV filter, fluidized bed, and heaters (if needed). A series of experiments were planned to examine larval cannibalism due to stocking density and/or feeding strategies that might help lower the costs of snook larvae culture. These studies included:

• a comparison of larval survival among tanks having different egg stocking densities;
• a comparison of larval survival in a green-water culture system using live algae versus larval survival in a system using algae paste;
• a comparison of larval survival in tanks with an increased live feed density (30 rotifer/ml), opposed to the standard rotifer density (15 rotifers/ml); and
• a comparison of larval survival with night feeding (at 10 p.m.) in addition to a normal daily live feeding schedule versus the addition of live feeds during the day only. The daily feeding schedule involved feeding rotifers volumetrically at 8 a.m., Noon, and 4 p.m.).

At total of 8 different batches of snook larvae were stocked in the experimental systems in the spring and summer of 2003. However, we encountered a number of problems and were unable to keep the larvae alive in the low-volume experimental systems. The longest a larval batch survived in the systems was 26 DAH (days-after-hatch) and the average survival among all the batches was 13 DAH. Possible causes of low larval survival were examined throughout the rearing season. Temperature was believed to be the most likely factor responsible for low larval survival. During 2003, it was not uncommon for the experimental larval systems to reach temperatures of 32oC. Fans were placed in the room containing the experimental systems in order to help reduce temperature during the summer months; however, the high temperatures continued throughout the snook larval rearing season. Therefore, the planned larvae experiments of the 2003 proposal were unable to be completed. Plans have been made to move these smaller volume experimental systems in 2004 to a location that will allow better temperature control during the study period.

Other culture strategies have been implemented in the MML live feeds department in order to lower snook larvae culture costs. Over the past years, live feeds production has remained the highest cost of snook culture. Live feeds have been provided to larval rearing systems at high densities (30 rotifers/ml), which resulted in high production cost. In 2003, we lowered the feeding level to15 rotifers/ml and were very successful at rearing larval snook in the production systems. Another cost-saving strategy that we are examining is the use of a new rotifer culture system. We initiated experimental production trials in summer 2003 with recirculating rotifer culture system (RRCS). Rotifer densities have been reported to reach as high as 2000-3000/ml in experimental RRCS systems with similar designs. To date, we have been able to increase production densities from 500-700 rotifers/ml in Batch Culture Systems (BCS) to 1500 rotifers/ml in RRCS for short periods of time. Using this estimated production density (1500 rotifers/ml), we could reduce our labor and production costs for live feeds by 50%.

Development of nursery culture methods for juvenile queen conch
New conch recirculating culture raceway systems were constructed at both MML’s Florida Keys locations, as well as Mote Aquaculture Park. Juvenile queen conch will be purchased from Harbor Branch Oceanographic Institution and nursery culture trials will be initiated in these systems restocking studies in the Florida Keys.

Larval Rearing Production Results
In 2003, the production results for fingerling snook have been very successful. At 3 DAH, the estimated total number of larvae produced was 554,400. At first size grading, juveniles were counted and 23,665 snook were transferred to nursery culture tanks. As of December, CARD had successfully reared a total of 13,800 juvenile snook.

Juvenile Diet Studies
A feed comparison study was conducted with snook fingerlings to evaluate commercial diets with different protein, carbohydrate and vitamin C content. This study is helping us determine the optimal diet for producing high-quality snook for stock enhancement research. CARD also worked with University of Florida and Walt Disney World veterinarians to detect potential skeletal deformities in juvenile snook (1-5cm) using radiograph technology. This study will help us determine the minimum size of juvenile snook to radiograph and accurately detect skeletal deformities.

Develop complex microparticles (particles made up of separate inclusion and carrier particles) capable of supporting growth and development in marine fish larvae (Subcontracted to University of Idaho; Investigators: Dr. Ronald Hardy, 3059F National Fish Hatchery Rd, University of Idaho, Hagerman, ID 83332 Dr. Mike Rust, Northwest Fisheries Science Center, 2725 Montlake Blvd. E., Seattle, WA, 98112)

In the 2003-2004 phase of this project, we are focused on the following two specific tasks.

1. To evaluate protein micro-sponges as inclusion and carrier particles
Protein microsponges will be evaluated as means of delivering LMWS nutrients to larvae. The digestibility of particle types that have satisfactory retention efficiencies for amino acids will be qualitatively determined in experiments with larvae using markers. Promising particle types will be evaluated in terms of leaching of soluble protein, larval feed acceptability and if resources allow, growth experiments.
Status: Experiments are currently in progress on this task.

2. To develop a method to determine apparent protein digestion and absorption efficiency for fish larvae and use it to evaluate promising microparticulate diets.

A sensitive quantitative method will be developed to determine dietary protein digestibility by fish larvae by comparing the ratio of protein to inert marker in the food and feces of larvae. The development of this method will provide us with a powerful tool to determine diet utilization and will greatly facilitate microparticulate diet development in the future.
Status: Experiments have been conducted with larvae of clown fish (Amphiprion perculaand are underway with larval rockfish (Sebastes sp). Analytical methods are now being adapted to quantify the micro quantities of protein and marker in larval fish feces. This task is on schedule.

Test the use of a combination vaccination/coded wire-tagging machine on a marine species
An automated tagging/vaccination machine developed by Northwest Marine Technology for salmonids was tested to determine if the equipment can be adapted for marine fish. The device relies on a fish’s natural behavioral traits like swimming into currents and choosing between different environments (lighted versus darkened areas, deep versus shallow water, etc.) to volitionally enter the system without anesthetic to be tagged and/or vaccinated.
Status: The system was successfully tested and adapted to use with Japanese yellowtail, Atlantic Cod, Pacific Cod and Yelloweye Rockfish. Data analysis and final report write-up are proceeding on schedule

ii. Develop optimal stocking strategies

Preparation for Pilot Scale Releases of Winter Flounder
We intend to release 10,000 juvenile winter flounder in the coming year. This experimental release will allow us to: 1) estimate the mortality rate of released fish, and compare it to wild fish; 2) estimate the growth rate of released fish, and compare it to wild fish; 3) describe the diet of released fish, and compare it to wild fish; 4) study the movements of released fish, and compare them to wild fish; and 5) gain insights about the carrying capacity of the release location. During the reporting period the juveniles we will use have been produced. We also applied for and received all necessary permits, and have made arrangements for assistance in field sampling with the New Hampshire Dept. of Fish and Game and Normandeau Associates.

iii. Evaluate the effectiveness of stock enhancement

Test of density-dependency effects with hatchery-reared juvenile snook released in critical nursery habitats - followup sampling
As followup work to continue the test of density dependence in juvenile snook, we conducted a standardized sampling effort during June 2003. This effort is described in the previous year reports and was conducted similarly. A total of 120 nets were deployed in four creek systems and all wild snook were tagged with visible implant elastomers (with different colors coding for each creek) and passive integrated transponder (PIT) tags. These data will be used to determine the relative abundance of the 2002 year class in comparison with previous years. In addition to other goals, the tagged yearling fish will help us track contribution rates of the different nurseries to the adult population.

Fishery dependent sampling of snook populations in sarasota bay: 6th Annual Snook Shindig
On October 3-4, 2003 Mote Stock Enhancement Researchers of the Snook Program and the Snook Foundation held the 6th Annual “Snook Shindig” Saltwater CPR (Catch Photograph and Release) and research fishing tournament to benefit the Snook Foundation at Mote Marine Laboratory. For the second year it was affiliated with the IGFA (International Game Fish Association). Unlike other years, however, the tournament was a qualifying event for the Rolex/IGFA Inshore Championship Series. The participants of the tournament was open to the public, including previous tournament anglers, friends of MML snook enthusiasts and local fishing guides. A $50.00 entry fee for adults and a $30.00 fee for youth was implemented.

The specific goals of the Snook Shindig were the same as the previous years tournaments and were to:

• To promote angler awareness and enthusiasm of the stock enhancement program, and the Snook Foundation’s research activities,
• allow stock enhancement researchers to further develop a working relationship with local fishermen,
• allow researchers to collect important dispersal information on the tagged and released snook in Sarasota Bay and its surrounding waters,
• allow researchers to collect important angling CPUE data of the hatchery snook,
• allow researchers to collect fishery contribution rates of hatchery snook from different areas of Sarasota Bay and its surrounding waters,
• allow researchers to collect growth and condition data from both wild and hatchery snook, and
• attain a relative contribution effect of hatchery-released snook to wild snook populations among years.

The Captain’s Meeting started at 5:30 pm on October3rd, with Chris Malkin (Snook Foundation Board Member) welcoming the participants to the tournament and explaining the tournament rules and point system (Appendix I). Dr. Ken Leber from MML was introduced and discussed the importance of stock enhancement and the data collected in the tournament. Bill Halstead from FWC/SERF followed with information on the special permits issued to each angler. Vicki Mooney, Roger Debruler, Nate Brennan, Jason Rock, Brett Blackburn, Meaghan Darcy and Dave Wilson from MML’s Stock Enhancement Program, and tournament volunteers were present. Over 70 anglers were present at the Captain’s Meeting. “Lines in the water” started at 7:30 pm October 3, 2003 and the tournament was officially over on October 4th at 12:00pm.

Special Authorization Permits - Special authorization permits were issued among the 73 anglers who attended the captains meeting. One permit was issued to each fishing “group” and allowed participating anglers to hold undersized snook until an official checked the catch. This permit was only valid for the duration of the tournament.

Results - During the tournament, 155 snook were caught and recorded by our weigh-in stations (Table 1). Three tagged snook were captured and of those 1 was a hatchery-reared snook (Table 2). Tagged snook were identified by the presence of a coded-wire tag (CWT).

The recaptured hatchery snook had been released in Bowlees creek on April 6, 1998 and ranged between 145-202 mm at release. The fish was recaptured off Longbar by Geoffrey Page and was 587 mm at weigh-in.

The CWT detectors used in this tournament were lent by the Florida Fish and Wildlife Commission in St. Petersburg and FWC/Serf facility in Port Manatee (5 upright detectors and 6 wands). Without these, the tournament would not have been possible.

iv. Develop habitat/release model for winter flounder (Primary responsibility: University of New Hampshire)
1. Gaining additional information on the habitat requirements of juveniles.
Winter flounder are certainly among the most thoroughly studied of all marine fish species. This wealth of information will facilitate the development of a winter flounder stock enhancement program, but further information on juvenile ecology and habitat is needed before optimal release strategies can be developed. Our most recent contribution to understanding flounder ecology, funded through SCORE, has been to use Habitat Suitability Index (HSI) modeling to predict appropriate release locations for winter flounder in the Great Bay Estuary of New Hampshire. Habitat variables used in the modeling included temperature, salinity, depth, substrate type, prey availability, and predator abundance. This highly successful study was completed in late 2002, and manuscripts are being prepared for publication.

2. Juvenile Production of Winter Flounder
Juvenile production began in March 2003. Adult winter flounder were obtained from local commercial fishermen and transported to the UNH Coastal Marine Laboratory (CML). Several groups, each consisting of 2 males and 1 female, were held in 1m³ circular tanks supplied with flowing seawater. Spawning occurred volitionally in these systems, and embryos were moved to 6m³ tanks supplied (1 liter/min) with filtered (5 micron), ultraviolet treated, ambient temperature (5-6°C) seawater. Three to four days after hatching, microalgae were added to the tanks each day, and the larvae were fed microalgae-enriched rotifers (Brachionus sp.) twice daily at a rate of 4000 per liter. This diet was replaced by DHA Selco™ enriched Artemia nauplii as the larvae increased in size. After the fish metamorphosed, weaning began by co-feeding enriched Artemia and the weaning diet (Biokyowa ™). Over the course of 10 days, the ratio of live food to dry diet decreased until the fish were only offered formulated food. As the fish increased in size, the weaning diet was replaced by a formulated diet produced by Nutreco (Gemma). Particle size increased as the fish grew. Approximately 30,000 juveniles (30mm) were produced.

3. Test the predictive capabilities of HSI models through in-situ experiments.
The Habitat Suitability Index models were used to identify 2 sites that, at least on the basis of substrate, temperature, prey availability, and predator abundance, seem to be quite suitable for winter flounder juveniles. We intend to test the predictive capabilities of this model in the summer of 2003 through in-situ experiments. In this work, we will test growth and survival at 5 geographically separate locations. Two of these will be the locations that the HSI modeling predicted as “best” in the Great Bay Estuary, and 2 will be locations the modeling predicted as “worst” in the Great Bay Estuary. The fifth location will be in the Hampton-Seabrook estuary at the proposed pilot-scale release location. Progress towards this objective have included the building of the 1m3 test enclosures and the production of the juveniles we will use in this study.

v. Develop adaptive management strategies

Assist the Florida Fish and Wildlife Conservation Commission with strategic planning for marine stock enhancement
In line with the short and long-term objectives of strategic planning for the Department’s marine stock enhancement program, several steps have been made toward (1) improving the effectiveness of the Department’s marine stock enhancement program, (2) adapting and refining the aspects of a “Responsible Approach to Marine Stock Enhancement” (Blankenship and Leber, 1995) that have not yet been fully implemented in Florida, and (3) incorporating adaptive management strategies into the agency’s stock enhancement program.

Dr. Ken Leber has been working closely with the Florida Fish and Wildlife Conservation Commission’s Stock Enhancement program to further our partnership in stock enhancement. Leber has continued to work closely in developing and implementing the pilot releases and nursery sampling of the juvenile red drum released in Tampa Bay. Leber also attended several staff meetings, and additional meetings with Bill Halstead, at the states stock enhancement research facility to assist as needed in planning and coordinating ongoing stock enhancement efforts.

vi. Identify released hatchery fish

Tagging Winter Flounder
All juveniles we have produced have been tagged with Coded Micro Wire Tags (CMWT) developed by Northwest Marine Technologies, Inc. Our previous tagging studies have shown that there is a size-specific mortality associated with coded wire tags. Fish < 21 (+/- 4.0) mm and 0.2 (+/-0.1) g are mortally wounded by the tagging process and /or the tags themselves, so we delayed tagging until the fish were greater than 25 mm. When the fish are released, a sample of 100 fish will be held in the laboratory to confirm tag retention. We also hope to use ultrasonic tags to monitor the small-scale (10’s of meters) movements of the released fish. Because ultrasonic tags small enough to place on fish as small as 25 mm do not currently exist, we are developing the technology in conjunction with a commercial ultrasonic tag manufacturer, Sonotronics.

Refining tag technology with the common snook
Currently we are making efforts to refine VIE tag capability for stock enhancement with juvenile snook. Previous studies at Mote Marine Laboratory with VIE in juvenile snook reported promising VIE retention in the caudal fin rays, while poor retention was reported in the head and jaw areas of the juvenile snook. This manuscript (Brennan, et al.) is entitled “Adapting tag technology for stock enhancement of the common snook, Centropomus undecimalus,” and is in revision to be published in the North American Journal of Fisheries Management. The paper describes results primarily obtained from field recaptures and lab studies detailing tag experimentation work from 1997-2002.

We have adapted PIT tags to juvenile snook for use in ecological studies testing density dependence. In these experiments, we tag wild juveniles throughout a nursery system according to size, microhabitat capture location, and date. Follow up large-scale releases will be performed in these systems and recapture efforts will determine the impact of large-scale releases on existing wild snook populations. We will look into the potential of developing and employing benign monitoring systems with these tags

vii. Describe life history patterns and ecological interactions

Determination of Sex Ratio of Winter Flounder
Studies have shown that sexual differentiation, and therefore male:female sex ratio, in some flatfish species can be influenced by juvenile incubation temperature. This also may be true for winter flounder, whose juveniles are quite eurythermal), but sexual differentiation and the sex ratio of cultured fish have never been investigated.

Because the sex ratio of cultured winter flounder, and the factors that may influence it, are completely unknown, and because the sex ratio of stocked fish is fundamentally important, we have begun to study sexual differentiation and cultured fish sex ratio as part of this study. We have sampled 30 fish from the general culture population at approximately 10mm total length (TL) intervals, starting at metamorphosis. Sampling will continue through the first year. Tissues have been fixed in modified Davidson’s fixative, and await histological processing. Slides will be examined to view structures and cells associated with gonadal tissue. By examining the size series of fish collected, we will be able to determine the size and age when sexual differentiation occurs, as well as the sex ratio of the cultured population.

Assessment of snook spawning distribution and frequency
A protracted spawning season has many biological implications such as multiple spawning efforts of individuals, energy allocation over a spawning season, changes in egg quality, and spawning site preferences. In testing the feasibility of a stock enhancement program for the common snook, development of a sound aquaculture system for snook is necessary and knowledge of snook spawning biology is an important aspect of this.

Eggs derived from different terms of the season may vary in hatching success and larval quality. An understanding of the ecological ramifications of these is important for responsibly producing progeny for year class supplementation.

Wild caught female snook were tagged with Passive Integrated Transponder (PIT) tags throughout the spawning season of summer 2003. Each female was scanned for previous tags, measured, aged (from scale annuli, aging only first time tagged fish), given an index of maturation, and the stage at which eggs are harvested. We categorized female maturation stages as follows:

Maturation Stage   Code
Unsure of sex          0
Female, but not ready          1
Female, almost ready          2
Eggs flowing          3
Spawned out          4

The stage at which eggs are harvested from mature females are assigned to codes as follows:
Eggs Harvested    Code
No Eggs          0
Eggs harvested 1st try          1
Eggs harvested 2nd try          2
Eggs harvested 3rd try          3

Snook were collected during spawning cycles on the new moon and full moon phases from May through September. Collections are conducted at spawning sites from Venice Inlet to Rattlesnake Key. Snook were captured with seines and trammel nets. All tagged fish were released after processing.

Evaluating cannibalism intensity in juvenile snook
Cannibalism is a common behavior among many piscivorous fishes and has been documented with the common snook. To responsibly test the feasibility of stock enhancement of snook, understanding the extent and intensity of cannibalism in juvenile snook, (similar to the sizes and ages of snook we are stocking) is necessary. If snook are highly cannibalistic during juvenile phases then stocking could have detrimental ramifications, such as hatchery juveniles significantly cannibalizing wild juveniles, wild juveniles cannibalizing hatchery released juveniles, or combinations of both. In any of the above cases, environmental systems will have a limitation on the amount of snook supported, and stocking activities may not be an effective means for replenishing a population.

During Fall, 2003 a size-specific cannibalism study was conducted with juvenile snook. This study was related to its potential effects on stock enhancement programs. Large enclosures were stocked with snook of different size combinations, densities, and with different alternative prey combinations. 20 enclosures (10’x12’ square) were established at two fundamentally different habitats (island habitat, and creek habitat). Snook were stocked in the enclosures and 1 week later, the remaining individuals were harvested. Week long trials were conducted in the enclosures for an 8 week period. We are preparing results from this study and will be included in the Final Report for this fiscal period.

References Cited
Blankenship, H. L. and K. M. Leber. 1995. A responsible approach to marine stock enhance-ment. In Uses and effects of cultured fishes in aquatic ecosystems. American Fisheries Society Sympo-sium 15:165-175.

Leber, K. M. 2002. Advances in marine stock enhancement: shifting emphasis to theory and accountability. Pp 79-90 In Stickney, R. R. and J. P. McVey (eds) Responsible Marine Aquaculture CABI Publishing, New York.