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 by SCORE scientists, 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 uncertainties 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 contract commenced in September, 2001. This interim report covers progress made during the period January 1, 2004 through June 30, 2004.

B. Project Accomplishments:
Mote Marine Laboratory Progress ­ January through June, 2004
Prior to the 2004 spawning season, Mote Center for Aquaculture Research and Development (CARD) staff members (Dr. Kevan Main and Michael Nystrom) visited the National Marine Fisheries Service Manchester Laboratory in Washington State to identify potential collaborative research opportunities and see the experimental culture facilities that are being used for behavioral and acclimation studies with marine stock enhancement species. An abstract and presentation on the culture of common snook has been submitted to be presented at the 2005 World Aquaculture Society conference in New Orleans.

Wild Strip-Spawning Efforts with Common Snook
The 2004 snook spawning season to produce juvenile snook for stock enhancement research began in late June. The spawning efforts were postponed this year because the release schedule for 2003 cultured snook was delayed until early June to conduct sonic tracking experiments with larger snook. In addition, the construction activities for new snook aquaculture facilities at Mote Aquaculture Park required that large number of staff were available for facility construction. In June, a two spawning attempts were completed. These spawning efforts were conducted to provide fertilized eggs to the Florida Fish and Wildlife Conservation Commission facility in Port Manatee. We also obtained a small number of larvae for egg quality studies at Mote. This collaborate effort produced an estimated 700 mls of fertilized eggs, which yielded approximately 139,460 larvae. These larvae were later stocked in a one-acre pond and a quarter-acre pond at the Port Manatee facility. Results from these stocking events are not currently available. July and August spawning attempts will concentrate on snook production for egg quality studies and production efforts at Mote. Excess eggs that are collected will be available for the Port Manatee research effort for further pond studies.

Development of a year round captive spawning protocol for common snook
In February, the first group of snook broodstock were transferred from Mote’s main campus to the Mote Aquaculture Park’s (MAP) new broodstock maturation facility. By late April, a total of twenty-four adult snook were transported and established in two independent tanks (54,315 L/tank). The fish are currently being held in freshwater while we continue construction of additional saltwater filtration systems. The broodstock maturation rooms are equipped with a light activation system that simulates sunrise/sunset and moon cycles. Temperature control systems are also installed on each maturation tank system. The snook will be monitored for egg development and growth every quarter.

Determination of an optimal diet for common snook broodstock
The captive broodstock at MAP continued to be maintained a fresh cut broodstock diet of shrimp, kapelin, squid, herring, and mackerel. These fish are monitored for egg development and growth every quarter.

Larval Rearing Production Results
In 2004, we continued to rear fingerling snook for the stock enhancement research effort until release studies were initiated in late May and early June. These snook were size graded every month. Growth and survival were monitored until the fish were released. Approximately 7795 juvenile snook were tagged and released in May and June 2004.

Development of nursery culture methods for juvenile queen conch
The first part of an experiment to evaluate the effect of stocking density and increased dietary calcium on growth rates of Florida queen conch was completed in June. This experiment is a collaborative effort with Harbor Branch Oceanographic Institution (HBOI) to optimize queen conch culture techniques and improve the success of restoration efforts. Higher growth rates were seen in the low density treatments. The diet study is evaluating the effect of additional dietary calcium on growth rates of juvenile queen conch. Early results indicate that there is no increase in shell length, but at Mote we saw a slightly higher weight in the conch that received the high calcium diet. This experiment is being continued by Mote for an additional six months at Mote’s Key West laboratory.

We are also rearing a group of Florida queen conch at our MAP site for stock enhancement research trials. These conch are now 30 mm average length and we estimate that they will need to be cultured for another six months to reach the appropriate size for release.

Test of density-dependency effects with hatchery-reared juvenile snook released in critical nursery habitats
In 2004, Mote’s Center for Fisheries Enhancement biologists will increase the abundance of wild juvenile snook in each of two treatment creeks by 400% and increase the abundance of wild juvenile snook in each of two control creeks by only 10%. Pre- and post-release sampling designs will be identical to our 2002-2003 study. Standardized sampling will occur prior to the release this summer, and after the releases in August-November 2004, and in January-March 2005. Releasing hatchery snook at 4-times the abundance of wild age-0 snook will allow us to determine if carrying capacity was reached in the treatment creeks.

As a further evaluation of abundance and distribution of juvenile snook before and after the releases, we plan to use acoustic telemetry technology in the juvenile fishes. Specifically the goals of the telemetry component of this study are to evaluate habitat use, survival, and interaction between the wild and hatchery reared individuals. We will tag wild and hatchery juvenile snook with sonic transmitters (Vemco, V8SC1L 24mm tags) prior to the releases in both control and treatment creeks. A total of 36 hatchery (9 per creek) and 36 wild snook (9 per creek) will be tagged and released with these tags. Within each creek, movements will be automatically recorded with submersed, semi-permanent acoustic receivers as well as active tracking activities with a hydrophone. Active tracking will be conducted over 24 hour periods, and diurnal habitat usage will be specifically recorded. Acoustically tagged snook will be tracked throughout the fall and winter (V8SC1L battery life ~ 300 d).

In April 2004, 7,737 juvenile hatchery-reared snook were tagged for this experiment and are currently held at the laboratory until release (Table 1). Tagging occurred at the MML Aquaculture facility. Kerry Mesner and Cindy Armstrong (FWC Stock Enhancement Research Facility [SERF] biologists from Port Manatee) assisted Mote staff during the tagging. Mark IV coded-wire tag machines and detectors on loan from SERF were used to tag the fish. All fish were additionally tagged with red VIE marks in the caudal fin as an external identification of a hatchery fish. Subsamples of snook from each system were subjected to health exams by an independent agency and were considered “healthy” for release. Releases are planned for the near future once all transmitters are successfully implanted in wild snook in each release creek.

An evaluation of cannibalism risk in juvenile snook
We propose to quantify allometric cannibalistic tendencies of age-0 and age-1 snook in a natural environment and in the laboratory. In the field, 10’x12’enclosures were stocked with snook of different size combinations, densities, and with different alternative prey combinations. Snook were held in the enclosures for 1 week after which all remaining snook and other prey species were harvested and counted. Losses of prey will be attributed to cannibalism/predation by the larger snook. We plan to conduct further enclosure trials this fall when more hatchery snook are available. Details of this study will be reported as progress is made.

UNH Progress ­ January through June, 2004
The overall goal of our winter flounder stock enhancement program is to accelerate recovery of the fishery by increasing spawning stock biomass. To meet this goal, we have developed a multidimensional research program designed to produce large numbers of high quality juveniles, to optimize release strategies, and to test the feasibility of winter flounder stock enhancement. Elements of the program addressed in this reporting period have included:

Rearing, tagging and releasing winter flounder juveniles
We have produced approximately 5,000 winter flounder for the 2004 release. These fish will be tagged in early August with Decimal Coded Wire Tags‘ and released into the Hampton-Seabrook Estuary. Prior to their release, 500 wild juvenile winter flounder will be caught at the release site, tagged with Visible Implant Alphanumeric Tags, and immediately released. Sampling for these released fish will begin the day after release and continue per methodology outlined in the grant. This experimental release will hopefully 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.

In late June, the remainder of the 2003 batch of cultured fish was released. These 562 cultured fish (mean size = 10.9 cm) were released in the Hampton-Seabrook estuary. These fish were tagged with a mixture of tags including, Visible Implant Elastomer Tags, Decimal Coded Wire Tags™, and Visible Implant Alphanumeric Tags. Releases were done by transporting the fish, via live tanks on a boat, to the release site. Here, divers transferred them into acclimation cages located on the bottom of the estuary. Forty-eight hours later, divers released the fish from the acclimation cages into the wild. Sampling for these fish has continued in conjunction with the 2004 characterization of the estuary. So far, no cultured fish have been recaptured.

Characterization of the Wild Winter Flounder Population in the Estuary
Because relatively little is known about the winter flounder population within the Hampton/Seabrook Estuary, we intend to increase our previous sampling regime both temporally and spatially. By doing this expanded sampling, we will be able to determine seasonal effects on 1) the abundance of winter flounder in the estuary, 2) the size class distribution of winter flounder, and 3) the spatial use of the estuary by different size classes of flounder.

Five sampling stations were selected throughout the estuary based on their physical and geographical parameters. At each station, a fixed submersible DST CTD data logger (Star-Oddi ™) was anchored to a cinder block and records temperature and salinity hourly. Surveys of these stations began in June and will continue through December 2004. Three types of collecting gear are used on each sampling occasion. In shallow water (<1.5m) we employ a 17m x 2m beach seine. Three replicate samples, each with an approximate swept area of 550m², are taken near low tide. In mid-depth areas (1.5-3m) we employ a 1m beam trawl. Three replicate 50m long tows are taken within the 1.5-3m depth interval, each parallel to the shore. In the deeper areas (>3m) we use a 4.8m otter trawl with 25mm mesh in the body and 6mm mesh in the cod end. As with the beam trawl, three replicate tows are taken within the depth interval, each parallel to the shore, and each approximately 100m length. The catch from all sampling is identified, enumerated, and measured. All winter flounder caught are checked for tags. Abundance is estimated as catch-per-unit-effort (CPUE), given as number caught per m² sampled.

To characterize the benthic community in the estuary and, therefore, winter flounder prey availability, we take a bi-weekly series of 6 replicate benthic cores (0.0079m² to a depth of 10cm) at each station throughout the duration of the sampling regime. Cores are stored in Zip-Lock™ bags, placed in ice, and returned to the laboratory where they are sieved through a 1mm mesh sieve. All prey taxa are stained with Rose Bengal and preserved in 10% buffered formalin and until they are identified, counted, and weighed.

Determination of Sex Ratio of the Wild Flounder Population
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 juvenile wild fish have never been investigated. While it is assumed that a 1:1 male to female ratio exists in nature, no one knows this for sure. Over the past 25 years, winter flounder populations have been declining while their natal estuarine water temperatures have been increasing. It is unknown whether there is a direct relationship between these two trends.

To make sure that the wild juvenile flounder population in the study site has an equal sex ratio despite rising water temperature, we intend to examine the gonads of 120 fish from the Hampton/Seabrook Estuary. This study will begin in August when we will randomly collect 24 fish from each of the 5 sampling stations. Fish will be collected in August because that is when we expect that they will be > 40 mm, and as such, easy to sexually identify histologically.

Sexual differentiation
Sexual differentiation of winter flounder has 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 are studying sexual differentiation and sex ratio of cultured fish as part of this study. Throughout the 2003 growing season starting at metamorphosis, cultured winter flounder were collected as they grew in 10 mm length increments, preserved, sectioned, and stained. Analysis of this study continues, and initial findings suggest that the sex of winter flounder as small as 40 mm TL can be determined histologically. This study will be completed by November 2004 and the results will be presented in December at the Flatfish Biology Conference (Westbrook, CT) and in January at the Aquaculture America Conference (New Orleans, LA).

Stress Physiology
A series of stress experiments have quantified the physiological effects of tagging, transportation and density on juvenile winter flounder through whole body measurements of cortisol concentrations. For the tagging experiment, juvenile winter flounder (42 mm mean total length + 0.1 SEM) were marked with either a visible coded wire (VIE) or decimal coded wire (DCWT) tag and allowed to recover in a separate holding tank. In order to determine the time period needed for cortisol to return to baseline values after tagging, each tagging experiment was divided into seven time trials. After the initial tagging (time 0), flounder were sampled every three hours for 12 hours (i.e at 3, 6, 9, and 12 hours) after the initial tagging occurred. Two additional samples were taken at 24 and 48 hours. The results from the tagging experiment indicate that VIE tags provoke a stronger and more prolonged (p<0.05) increase in cortisol levels when compared to decimal coded wire tags (DCWT). For the transport and density studies, the effects of stress at five different stocking densities (100%, 200%, 300%, 400%, and 600%) were investigated during transport at four different transport intervals (5 minutes, 45 minutes, 90 minutes and 48 hours). Transport produced a stress response at all densities 45 minutes and 90 minutes after transport. However, only fish stocked at a density of 600% sustained cortisol levels that were significantly different than control levels after 48 hours. The results of this study suggest that regardless of transport density or tag type, a minimum of 48 hours are needed for the juvenile flounder cortisol levels to recover to baseline/control levels. This information has been submitted and is currently under peer review for publication.

Northwest Fisheries Science Center Progress ­ January through June, 2004

Task 1 Larval Diet Development
Larval digestibility methods development
A talk was given at World Aquaculture 2004 in Hawaii to highlight progress on the uptake of inert digestibility markers by Artemia nauplii and rotifers. The three main objectives of the trial were: To determine if rotifers and Artemia can take up Yttrium, Ytterbium, Dysprosium, and Lanthanum oxides; Determine the retention of markers over time to understand how much marker will fish actually be consuming; Determine if markers affect survival. Rotifers and Artemia nauplii took up all markers in quantities sufficient for analysis (up to 20% of the organisms dry weight) within 15 minutes. All markers depleted from Artemia nauplii over time. A significant drop in marker concentration occurred between 1 and 15 minutes and 30 and 60 minutes (Figure 1). This drop in concentration could influence apparent digestibility coefficients if fish are not sampled quickly after feeding. Rotifer survival (93 + 5%) was reduced by the Yttrium oxide marking process but was not reduced by the other three markers. Artemia survival was reduced during the marking process by Yttrium (88 + 8%), Lanthanum (91 + 6%), and Dysprosium (88 + 3%) but not by Ytterbium oxide. Trials to determine depletion of markers from rotifers are being currently being conducted. We also determined that because some artificial diets loose soluble material quickly in the water causing marker concentrations to increase over time. The concentration of marker in the feeds on-the-shelf may be less than the concentration of the diet in the water.

Feeding trials were conducted at Manchester in March and April using Pacific cod larvae. Three groups of larvae (three tanks per treatment) all from the same parental cross, were fed either Yttrium marked rotifers, Yttrium marked Artemia nauplii, or a microparticulate diet containing yttrium developed in our lab (details below). Live feeds were marked using methods developed in the study mentioned above. On non-sampling days, all tanks were fed a combination of enriched (DHA/EPA) rotifers and Artemia nauplii several times a day. Treatment feeds were fed on days 30, 37, and 44 post hatch. Food was withheld from fish 24 hours prior to introduction of experimental feeds to ensure non-marked feeds were not present in the gut. Larvae were allowed to feed to apparent satiation (about 1 hour). Larvae in the microparticulate treatment ate much less than larvae from the live feed treatments. Feeding behavior of live feeds does not appear to be negatively influence by the Yttrium marker. Full fish were removed from the tanks and placed in scintillation vials (5 fish per vial) containing filtered autoclaved seawater. The vials were placed in a water bath and the larvae were allowed to evacuate for 18 hours. Larvae were removed carefully from the scintillation vials and the resulting feces samples were frozen at -10°C for later protein and marker analysis. Pacific cod have proven to be very hardy for feeding trials. They respond well to artificial and live diets, can be stocked at high densities, and are robust enough to endure handling stress and 18 hours in scintillation vials. We also performed a digestibility trial using rotifers marked with either Yttrium, Lanthanum, Dysprosium, or Ytterbium oxide to see if apparent digestibility coefficients differed among the marker treatments. The feeding trial was successful, however, the samples have yet to be analyzed

In order to evaluate the apparent digestibility coefficients (ADCs) of experimental microparticulate diets and the live diets fed to the cod (described above) methods have been developed to determine the concentrations of protein and Yttrium oxide (an inert digestibility marker) in the extremely small samples of larval marine fish feces. Similar to digestibility trials involving larger fish, the ADC of each diet will be determined by contrasting the ratio of marker to protein in the diet to that in the feces. Yttrium oxide has been selected as the inert digestibility marker due to its success as a marker in larger fish digestibility experiments and its high sensitivity when analyzed by inductively coupled plasma ­ optical emission spectroscopy (ICP-OES). We are currently able to quantify yttrium in larval fecal samples down to 0.8 ug. At this level of sensitivity, we have been able to quantify yttrium in the fecal samples from 2-week-old clownfish when feed a diet marked with 2% (w/w) yttrium. In April 2004, we applied the technique to analyze fecal samples from 3-week-old Pacific cod larvae. We successfully quantified marker in all but one group of samples, which corresponded to a replicate tank where a low incidence of feeding was observed.

Because of the small amount of fecal material that is recovered from larval fish feeding studies, conventional methods to determine protein (i.e. Kjeldahl and Dumas) are inadequate and a new method is needed. In the past, we have adapted a spectrophotometric protein assay method, the bicinchoninic acid or BCA protein assay method, to quantify digested proteins in a salt-water matrix. The detection limit for this method, however, proved inadequate and we were unable to quantify protein in fecal samples of 2-week-old clownfish larvae. During early Spring of this year we worked to lower the detection limit of the technique by concentrating reagents and incorporating alternative buffers which improved the solubility of some interfering magnesium compounds. Initial laboratory results with the microparticulate feeds indicate the modified method should be able to detect protein levels down to 20 ug. This is an order of magnitude reduction in detection limit from the previous method. In April 2004, we applied the new method to analyze fecal samples from 3-week-old Pacific Cod larvae and successfully detected protein in almost every sample. Results of this study are still being tabulated

Microparticulate diet leaching trials
Our work has demonstrated that it is possible to measure the leaching loss of protein from micro-particles in real time and fit the data to a natural decay equation very accurately. The equations can be used to predict protein loss at any time up to ten minutes from adding a micro-particulate formulation to water. The absolute mass of leachate solids was also measured. This method gives solid information on the potential efficiency of payload transfer of nutrients to the larval fish gut. Two Japanese commercial feeds and one NWFSC experimental feed were characterized to form a baseline reference. It is now possible to take any micro-particulate feed and reference its performance against the baseline feeds. More feeds are needed to add to this reference set to build a picture of properties which can be manipulated to increase micro-particulate performance.

Microparticulate diet development
A new experimental feed is being made that will incorporate low molecular weight trypsin digest and high molecular weight BSA. This will be tested by above method and the leachate will then be measured to look for differential protein loss of low and high weight protein fractions. This feed will also be fed to clown fish (or other species) and feces analyzed for digestibility.

Task 2 - Lingcod tagging study
No work has been conducted on this task during the first six months. We are scheduled to tag fish for release on August 2-3rd with releases about 2 weeks later. This task is on schedule and will be addressed over the next few months.

Task 3 ­ Pacific Cod tagging study
In February 2004 we collected 11 adult Pacific cod off of Blake Island in central Puget Sound. This was done in collaboration with WDFW on a chartered trawler. Efforts to collect by hook and line in the same area were unsuccessful. Of the 11 captured cod, 5 were females. Three of these successfully spawned in March. Another female cod captured last season and held for a year in captivity in the same area was also spawned at the same time. Fish were held on chilled water at 7 °C until spawning.

Eggs from the 4 females were incubated in two incubator types. The first type was a modified salmon egg incubator. These are down-welling incubators and approximately 4 liters in volume. The other incubators used were McDonald jars. These incubators are 7 liters in volume and provide an upwelling flow pattern. Both types of incubators proved successful for Pacific cod eggs.

Several hundred thousand larvae hatched from the eggs of the 4 females. A portion of the larvae were reared in two separate systems, 200 l tanks and 9300 l bags. Larvae reared in tanks were fed rotifers and Artemia while larvae in bags were fed wild zooplankton and supplemented with rotifers and Artemia. Larvae reared in bags grew faster. Additional Pacific cod larvae were used in the digestibility study as previously reported

Approximately 2000 Pacific cod survived through metamorphosis. They are currently being reared in net pens and will be used in a radio tag study when they are large enough to tag this fall.

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.