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 have partnered 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 stocking as a marine enhancement management tool, 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, 2004), 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.

Multi-Year Contract Period and Relation to this Reporting Period
This Multi-Year contract commenced on July 1, 2004 for the 5-year period ending June 30, 2009. The funding period for this 2nd year of the Multi-Year contract is July 1, 2005 through June 30, 2006. This interim report covers progress made during the period January 1, 2006, through June 30, 2006.

Project Accomplishments:
Mote Marine Laboratory Progress ­ January through June 2006
Aquaculture Research to Develop Rearing Technology for SCORE Species:
Wild Strip Spawning Efforts with Common Snook
The 2006 snook spawning season to produce juvenile snook for release-recapture experiments commenced in June. Collecting trips were conducted in mid and late June. Eggs and milt were collected and maintained separately in order to allow for subsequent genetic analyses of progeny from each mated pair. Fin clips were collected from each brood fish and shipped to Michael Tringali at Florida Fish and Wildlife Conservation Commission for microsatellite DNA (genetic fingerprint) analysis. Fertilized eggs were stocked into multiple larval rearing tanks. This work was underway at the end of this reporting period.
Controlled Maturation and Spawning of Common Snook
Maturation Trials
From January through June 2006, we continued our research to mature and spawn captive snook at Mote Aquaculture Park (MAP). Three large tanks (54,315 liters or 14,350 gallons per tank) were stocked with mature adult snook and two of those tanks (20-1 and 20-3) were included in a trial to manipulate temperature and photoperiod to induce maturation. Tank 20-1 was stocked with 15 fish (8 male:7 female) and tank 20-3 was stocked with 13 fish (5 male:8 female).

In November 2005, temperature and light conditions were adjusted to simulate a winter light and temperature cycle (10 h light:14 h dark; 22 + 0.5°C). Winter conditions were maintained for two months in both tanks, followed by an immediate temperature and light adjustment to tank 20-1 and a gradual adjustment to tank 20-3 (12 h light:12 h dark; 24 + 0.5°C) to simulate spring environmental conditions. One month later, we continued the gradual and immediate adjustments to simulate a summer photo-thermal cycle (14 h light:10 h dark; 30 + 0.5°C).

One month after the fish were conditioned to the summer cycle (their natural spawning season), the snook were biopsied to assess the state of gonadal maturation. Oocyte samples were obtained from 9 females ranging in size from 51-154 _m. The males were not freely expressing high volumes of milt; however, we were able to obtain small samples by manual expression or cannulation from 11 males.

One month later (May 2006), we sampled both tanks again and found oocyte diameters ranging from 341.9-473.4 _m in 3 females. These females were injected with slow releasing GnRHa implants at a dose of 50 _g kg^-1. All hormone implants were provided by the University of Maryland’s Center of Marine Biotechnology and were administered according to the methods described by Zohar et al. (1996). In May, the males continued to produce very little milt. Samples were obtained by manual expression or cannulation from 6 males.

In June, we sampled both tanks again and found oocyte diameters ranging from 341.9-447.1 _m. Eight females were implanted with GnRHa (3 females in 20-1 and 5 females in tank 20-3). Milt production improved in the June with samples obtained from 11 males.

Spawning Trials
Following implantation, the fish were allowed to spawn naturally in the tanks. Eggs were passively collected using an egg collector that skims floating eggs of the surface. In May, a small spawn was collected in both 20-1 and 20-3 three days post implantation. Approximately 5000 eggs were collected from both tanks and fertilization and hatch rates were estimated at 100% and 98.6% respectively. In June, we collected eggs from tank 20-3 on days 2, 3 and 6 post implantation. Fertilization ranged from 11.3-80.7% and hatch rates could not be estimated because larvae began hatching in the egg collectors. Larvae were stocked in experimental (3.5 L) and production (3300 L) tanks and were fed rotifers and copepods (Acartia tonsa). Larval trials were terminated 11 DAH due to water quality issues.

Studies at Mote Marine Laboratory To Develop Effective Release Strategies and Assess The Actual Effectiveness And Potential Impact Of Stock Enhancement:
Evaluate Snook Stock Enhancement Impact in Sarasota Bay and Tampa Bay
Refining release strategies to improve survival of released snook.
On 30 January 2006, we submitted a manuscript entitled “Predator-free enclosures improve post-release survival of stocked common snook” by Nathan P. Brennan, Meghan C. Darcy, and Kenneth M. Leber. This manuscript was published in June in the Journal of Experimental Marine Biology and Ecology Volume 335:302-311..

Test of Density-Dependency Effects with Hatchery-Reared Juvenile Snook Released in Critical Nursery Habitats

Proposed work in this area has been completed. We are currently working on a manuscript. We are presenting these results at the 3rd International Symposium on Stock Enhancement and Sea Ranching, which is in Seattle, WA, in September, 2006.

Refining Tag Technology with the Common Snook and Red Drum
Adapting Tag Technology toward Stock Enhancement of the Common Snook
We are investigating the use of acoustic transmitters (Vemco, V8SC1L, 24 mm long) in juvenile (age-0) snook. Both hatchery and wild snook were implanted with transmitters to monitor movement patterns in 2004 and 2005. We are currently analyzing the results of this study.

Adapting Coded-wire Tags to “Phase-I” Red Drum
A manuscript that describes these activities is in progress.
Feeding Ecology of juvenile snook:
Experiment 1: Gastric evacuation rates of finfish and shellfish prey items consumed by juvenile snook.
May-June, 2006. We are conducting a laboratory based study to evaluate gastric evacuation rates of different prey items consumed by juvenile snook. Wild snook are currently being collected and acclimated in the laboratory prior to the feeding trials. We will perform the PGL technique mentioned above to examine the stomach contents at pre-determined time periods after consumption. These results will validate the above mentioned study on snook feeding periodicity.

Fishery Independent Assessment of Adult Habitat
Identify Recruitment of Hatchery Snook to the Adult Populations
We produced a manuscript entitled “Investigations of essential fish habitat using releases of cultured snook.” The manuscript will soon be submitted to a scientific journal for peer review. In this study, we cross examined our results (survival and growth) obtained from short-term sampling in juvenile habitats with results from adult snook habitats obtained over the long-term (1-8 years after release).

Fishery Dependent Sampling of Snook Populations in Sarasota Bay
9TH ANNUAL “SNOOK SHINDIG”
Work has been underway to solicit sponsorships for the 9th Annual Snook Research Roundup Tournament and Shindig BBQ to be held October 20-21, 2006. This tournament is part of an annual long-term evaluation of stocked hatchery-reared snook contributions the snook fishery in Sarasota Bay.

An evaluation of cannibalism risk in juvenile snook
Results from this study are being analyzed and will be reported on when complete.

Assist the Florida Fish and Wildlife Conservation Commission (FWC) with Strategic Planning for the FWC Marine Stock Enhancement Program
In line with the short and long-term objectives of strategic planning for the Fish and Wildlife Conservation Commission’s marine stock enhancement program, several steps have been made toward (1) improving the effectiveness of FWC’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) identifying and prioritizing potential marine fish species for stock enhancement in Florida.

• Dr. Ken Leber (Mote Marine Laboratory) has been working closely with the Florida Fish and Wildlife Conservation Commission’s Stock Enhancement program as a chief advisor and co-leader in strategic planning and research planning in this program.
• Dr. Leber has continued to work closely with the state in developing and implementing the pilot release experiments to evaluate the effectiveness of releasing juvenile red drum in Tampa Bay to boost red drum population size there (Project Tampa Bay).

In addition to managing collaborative aspects of this project at Mote Marine Laboratory, Dr. Leber participated in several planning meetings with FWC staff during this grant period. These included meeting with

University of New Hampshire Progress ­ January through June 2006
The long-term 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, to understand how habitat attributes affect movement, and to study variables that may affect sexual differentiation of juvenile winter flounder. Elements of the program addressed in this reporting period have included:

Juvenile Fish Production:
We produced approximately 30,000 winter flounder from a wild-caught broodstock at the Coastal Marine Laboratory (CML). Currently 4,000 of these fish are being used in two laboratory experiments, while the remainder will be used for the final acclimation cage/predator study later in the summer. In addition, we maintained fish from the 2005 hatchery production. These one-year old flounder will be used for the telemetry studies in late summer/early fall.

Acclimation Cages/Predator Study:
Juvenile flatfish are vulnerable to a suite of predators, including many decapod crustaceans. For juvenile winter flounder, predation by green crabs Carcinus maenas is of special concern (Fairchild & Howell 2000). In a field study done prior to the 2004 release of fish, we found that average green crab density at the release site was 0.6 crabs/50 m². This density was not significantly different than adjacent areas. Our acclimation cages, stocked with cultured flounder, were then placed at the site so that the fish could adjust to their new environment for 2 days, and then be released from the cages. Within 4 days after the release, crab density increased 7 fold to 4.3 crabs/50 m² in the immediate area. Crab density returned to pre-release densities quickly thereafter, but cultured winter flounder density also decreased quickly. No cultured flounder were recaptured outside of the immediate release site despite frequent sampling. The sharp increase in crab density, combined with the sharp decrease in flounder, leads us to hypothesize that crabs may be attracted to, and aggregate around, the acclimation cages containing the fish, and that crab predation may have been responsible for the decrease in the cultured flounder density. Thus although acclimation cages are a necessary tool that allows the stocked fish to adjust to their new environment, they also may be a detriment if they attract predators to the site.

Last year, we conducted two of three field experiments to study the relationship between acclimation cages and predators. The first experiment determined if green crabs are attracted to the acclimation cages at the release site. Crab densities were determined by trawl surveys for 2 days. Following this, four acclimation cages were placed on the bottom at the release site. Two replicates contained flounder while two were empty (control). Surveys of crab density (#crabs/m²) within 5m of each cage were conducted daily for 3 days by SCUBA. Results showed that after one day, crab abundance was significantly (t-test, p < 0.01) higher on cages containing fish than on empty cages, proving that acclimation cages containing flounder do attract green crabs.

The second experiment determined if acclimation cages, even in the absence of fish within them, attract crabs because they provide structural relief in an otherwise relatively featureless environment. In this, a second site (control) was established 250m downriver from the release site. At both sites, crab densities were determined by trawl surveys for 2 days. Four empty acclimation cages were deployed at the release site while no cages were deployed at the control site. At the release site, surveys of crab density (#crabs/m²) within 5m of each cage were conducted daily for 3 days by SCUBA, while at the control site, trawl surveys were continued. Results showed that within 30 minutes of cage deployment, crab densities significantly increased in the release area (t-test, p < 0.01) and continued to increase each day indicating that green crabs are attracted to the empty acclimation cages.

Although cages are necessary for acclimating cultured flounder (Fairchild and Howell 2004; Sulikowski et al. 2005), they also are detrimental by attracting predators to the release site. Modification of the release strategy is necessary to offset this problem and alternate release strategies are being investigated. For example, in addition to our “standard” release protocol, we also will release acclimated flounder into a secondary release site where crabs have not aggregated and then we will compare crab density at both sites. The completion of this objective will be done later this summer.

The results from these acclimation cages studies were presented in May 2006 in Florence, Italy at Aqua 2006. The final experiment will be presented in September 2006 in Seattle, WA at the Third International Symposium on Stock Enhancement and Sea Ranching.

Temporal and spatial distribution of juvenile wild and cultured winter flounder in the estuary:
The objective of this study is to identify the areas within the estuary where juvenile fish are found, to characterize their habitat, and to study their temporal and spatial use of the estuary. In addition, cultured and wild juvenile movements will be compared. To accomplish this goal, both wild and cultured juvenile winter flounder were anesthetized, fitted with acoustic tags (VEMCO V7-2L-R256 coded pinger tags), and released. Each acoustic transmitter emits a distinctive coded pulse (frequency 69 khz) that is detected by a hydrophone, thereby allowing the fish’s location to be accurately determined, and the fish’s movements to be tracked over time.

Simultaneous releases of cultured and wild one year old winter flounder occurred in the Hampton-Seabrook Estuary, New Hampshire, USA. Wild fish were caught by beam trawl at the release site (Hampton River) and brought back to the CML where they and the cultured fish were fitted with acoustic tags. All fish were released 48 hours later by gently placing them into the river. Tracking began one hour after the fish were released and continued daily until either the fish could not be found or we suspected that the tag died. Fish were located manually using an omnidirectional and a directional handheld hydrophone. In order to describe fine-scale movements of juvenile winter flounder, a VEMCO VRAP system which automatically tracks tagged fish movements within a triangular array of buoys was deployed in the release area. This release on 28 November 2005 consisted of 2 wild fish (44.3 + 36.6 g; 151 + 49 mm) and 6 cultured fish (60.3 + 31.6 g; 153 + 18 mm), 2 of which had been acclimated in a cage (AC) for 48 hours. This group of fish was tracked for a total of 74 days until 10 February 2006. Though the receivers recorded continuously, the coded tags only pinged every 1-2 minutes. As such, there were times when the signals coincided simultaneously and the receiver could not distinguish the tags. Therefore, periodically fish were not located by the VRAP system even if they were within the range of the buoys. Also, due to the physical parameters of the narrow channel and the large tidal flux, we could not stretch the buoy triangle out very far. The fish did not, for the most part, remain within the VRAP range, and so we manually moved the receiver buoys so that we were tracking at least 2 fish at all times. This means that there is limited information on most of the fish released.

Non-acclimated cultured fish followed a similar pattern in which they quickly left the triangulation area. Within the first day, 3 out of 4 fish went downriver on the falling tide. The fourth non-acclimated cultured fish went upriver out of range of the VRAP system on the first day after release, then returned to the release spot 3 days later, and proceeded downriver. After this, these non-acclimated cultured fish were never located again.

Although both AC fish left the triangulation area too, it appears that they did not emigrate as far as the non-acclimated cultured fish. One AC fish went downriver while the other went upriver. We chose to track the one (fish 149) that went upriver since a wild fish was also nearby and we could then compare their movements. Interestingly enough, the fish that went downriver was located later at the acclimation cage site.

The 2 wild fish remained in the general area throughout the entire tracking period. Fish 144 was tracked regularly for the first 16 days but then headed downriver. This fish did move back in and out of the triangulation area later in the tracking period but we didn’t get as many fixes on it as the other wild fish 145. Fish 145 was tracked fairly reliably throughout the entire period and we used it as a comparison to the acclimated cultured fish.

Though not statistically possible, wild fish 145 and AC cultured fish 149 were compared for possible trends. Generally, the cultured fish moved more frequently but shorter distances than the wild fish. Kernal analyses of home ranges showed that the AC cultured fish maintained a much smaller home range than the wild fish both in the 50% probability (AC=9 m²; W=131 m²) and 95% probability areas (AC=97 m²; W=478 m²). Minimum convex polygon analyses corroborate this home range pattern (AC=723 m²; W=1683 m²). Data are still being analyzed to determine if there is a relationship between fish movements and tidal stage.

Results from these tagging studies were presented in May 2006 in Florence, Italy at Aqua 2006 and will be included at the annual meeting of the American Fisheries Society in September 2006 in Lake Placid, NY.

More telemetry work is planned for later this summer. We will finish our studies that were designed to identify the areas within the estuary where juvenile fish are found, to characterize their habitat, and to study their temporal and spatial use of the estuary by tracking fish manually and with VR2 submersible receivers.

Sex ratio of juvenile winter flounder:

Completion of previous studies
Wild winter flounder
We have shown that the sex ratio was significantly different than 1:1 in the 2003 cultured winter flounder population (males dominate 2.5:1)(Fairchild et al., In Review), and we know from studies with other flounders that water temperature can affect gender during differentiation. Because winter flounder spawn and develop throughout estuaries, which often have spatially different temperature regimes within them, it is possible that the sex ratio of wild fish varies from one part of an estuary to another, dependent on which part of the estuary they develop in. To investigate this, 3 sites in the Great Bay Estuary that differ in temperature were chosen for this study. Because winter flounder spawn in March/April, and an individual’s sex is determined at <41mm TL (Fairchild et al., In Review), we assumed that in the fish we collected, gonadal development would have been influenced by the thermal regime they experienced from hatching through gonadal differentiation. Temperature recorders were deployed to document temperature and a total of 89 one-year old juvenile winter flounder were collected by trawl in early June 2005 (age 1 fish) and 29 in late October 2005 (young-of-the-year). All fish were measured, euthanized with an overdose of MS-222, and preserved in Davidson’s modified fixative. Gonads were dissected out of each fish, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Slides were numerically coded and examined by three viewers in a blind test to determine sex based on structures and cells associated with gonadal tissue. Fish were scored male, female, unknown (gonadal tissue visible but unidentifiable to sex due to poor sectioning), or missed (no gonadal tissue present on slide due to poor dissection).

Although the data (Table 1) suggest that the age 1 fish collected from Little Harbor were predominantly female (_2= 14.96, df=1, p<0.01), 42% of the fish were not sexually identified due to poor gonad dissections. If all of these fish had been identified as males, then the population would not have deviated from a normal 1:1 ratio (_2=0.77, df=1, p=0.38). Therefore, these data should be looked at with great caution; we can not rule out that a normal sex ratio exists in Little Harbor (the coldest location). No differences in sex ratio were found at the other 2 locations for age 1 wild juvenile winter flounder (Table 1).

YOY wild juvenile winter flounder were only caught in Little Harbor during the fall 2005 (Table 2). Data are not available for YOY wild winter flounder from Broad Cove or Great Bay. After repeated failed attempts in fall 2005 to catch sufficient numbers of YOY fish in these locations, we ended this study. There was no difference in the sex ratio of the YOY fish collected from Little Harbor (_2= 0.15, df=1, p=0.69).

While wild juvenile fish analyses were interesting, one could argue that there is no way to know if these fish occupied the waters where they were caught during the ontogenetic period of gonadal development and differentiation. More noteworthy is the overall equal sex ratio of the wild fish in the Great Bay estuary (Table 1).

Cultured winter flounder
We also continued the sex ratio research on cultured juvenile winter flounder by (1) identifying sex of other year classes of cultured fish and (2) by manipulating juvenile rearing temperatures.

From the 2004 and 2005 cultured populations, a total of 149 and 50 fish were sampled, respectively, for sex identification. These fish were processed and analyzed in the same manner as the wild fish, with the exception of 117 fish from the 2004 cultured population that had died for unknown reasons but were preserved in Davidson’s modified fixative anyway. However, the sex of these fish was not identifiable due to the necrotic state of the tissue and therefore these fish were excluded from the analyses. For the 2004 cultured flounder population, a total of 17 females and 15 males were identified, yielding a sex ratio that did not deviate from a “normal” 1:1 ratio (_2= 0.12, df=1, p=0.724). This sex ratio was also true for the 2005 cultured population in which 23 females and 22 males were identified (_2= 0.02, df=1, p=0.881).

To determine if any thermal differences existed between years (2003-2005) which might explain the observed sex ratio difference in 2003, fish rearing temperatures was analyzed. If winter flounder exhibit TSD, then temperature would influence phenotypic sex prior to and during the ontogenetic development of the gonads. Gonadal differentiation in winter flounder occurs in fish < 41 mm TL (Fairchild et al., in review), and for this NH population that developmental period corresponds to fish < 110 dph. Therefore, if winter flounder exhibit TSD, temperature influences on sexual development are most critical between April and the beginning of August. Monthly rearing tank water temperatures for these critical months were compared for each flounder year class to determine if temperature affected the sex ratio of the 2003 population. Although significant differences were found in May, June, and July (Fig. 1), there was no clear pattern.

The mean rearing temperature for the 2003 population only was significantly different from the other two populations in July when it was 1.3 °C cooler on average (ANOVA, df=2, p<0.001). Slight deviations in temperature from year to year were expected at the CML since winter flounder are reared in a flow-through system using ambient temperature water which fluctuates both tidally and seasonally. However, it may be possible that an extreme temperature event (hot or cold), however brief, could affect the sexual differentiation in fishes that exhibit TSD if the thermal event coincided with some critical stage in the gonadal development. CML water temperature was never considered problematic if < 18 °C (Casterlin and Reynolds 1982) because winter flounder are eurythermal (Pereira et al. 1999).

To determine if winter flounder exhibit temperature-dependent sex determination (TSD), a temperature manipulation experiment was conducted in the laboratory with young-of-the-year cultured fish. Three replicates of 3 temperature treatments (5, 10, 15°C) were set up in climate-controlled rooms at the UNH. Experimental units consisted of glass aquaria filled with 40 liters of filtered, UV sterilized sea water. Each of these static systems contained a carbon filter, aerator, gross particulate sponge filter, and were illuminated from above (24L:0D). Fifty cultured fish (59 dph; mean TL 16.6mm) were stocked into each tank on June 20, 2005. Fish were monitored daily for 72, 123, and 86 days for the 5, 10, and 15°C treatments, respectively. Daily protocol included feeding (Gemma 300-500microns), and measuring water temperature, dissolved oxygen, salinity, and total ammonia. Twenty liters of water in each aquarium were changed approximately weekly, and total ammonia levels never exceeded 1 ppm. Too few fish survived in the 5°C treatment to generate meaningful results. In the other 2 treatments, once the fish were > 41 mm TL and gonadal differentiation was complete (Fairchild et al., in review), all fish were processed.

For the 10 degree treatment, a total of 12 females and 22 males were identified, yielding a sex ratio that did not deviate from a “normal” 1:1 ratio (_2 = 2.94, df=1, p=0.09)(Table 3). This sex ratio was also true for the 15 degree treatment in which 14 females and 16 males were identified (_2 = 0.13, df=1, p=0.72). These results suggest that 10 and 15 °C rearing water does not affect the sex of 59-123 dph juvenile winter flounder. In addition, there were no significant differences in sex ratio between the two treatments (ANOVA, df=1, p=0.24).

New laboratory experiment #1
Because we still do not know if winter flounder exhibit TSD, we are repeating the temperature controlled experiment using higher temperature treatments (10, 15 and 20°C). These span the range of temperatures experienced by post-metamorphic wild juveniles, as well as the range of temperatures used during the early juvenile stage in most winter flounder culture facilities (Howell and Litvak 2000). Again, these treatments have been set up in climate-controlled rooms at the UNH. Experimental units consist of glass aquaria filled with 40 liters of filtered, UV sterilized sea water. Each of these static systems contains a carbon filter, aerator, gross particulate sponge filter, and is illuminated from above (24L:0D). Fifty cultured fish (69 dph; mean TL 17 + 3 mm) were stocked into each tank on June16, 2006. Fish are monitored daily which includes feeding (Gemma 0.3 mm), and measuring water temperature, dissolved oxygen, salinity, and total ammonia. Twenty liters of water in each aquarium are changed approximately weekly so that total ammonia levels never exceed 1 ppm. Once the fish are > 41 mm TL and gonadal differentiation is complete (Fairchild et al., in review), all fish will be processed for sex determination and analyzed using Chi-square tests.

New laboratory experiment #2
The basic techniques for culturing winter flounder have been developed, and are reviewed in Howell and Litvak (2000). Nevertheless, there are issues relating to culture technology that need to be addressed. The first is a further investigation of juvenile stocking density. We have occasionally recorded high incidences of fin erosion, in which fraying of the fin edges, and erosion of the fin rays has occurred. Fin and tail erosion in juvenile winter flounder has also been observed by de Montgolfier et al. (2005). Although we have not yet determined if this condition affects the swimming or burying abilities of the fish, it is a possibility. Given this, and given that cultured fish released into the wild should not be disadvantaged because of morphology or behavioral differences, we believe it is important to explore the issue of fin erosion.

In an earlier study we examined the effects of juvenile winter flounder stocking density on growth, survival and behavior (Fairchild and Howell 2001). We explored the possibility that high stocking densities (up to 300% fish area to tank bottom area) caused an increase in fin nipping, and thus erosion of caudal fin area. Although there were no differences between treatments, ranging from 50-300% stocking density, it was clear that the smallest fish in all treatments had badly damaged caudal fins. We concluded that a size hierarchy had been established in all tanks, and that the smallest fish had suffered from the aggressive behavior (nipping) of the larger fish. Thus one possible explanation for the observed fin erosion is fin nipping associated with aggressive behavior. We know from some of our previous work that winter flounder juveniles held at high densities, even for short periods of time, display elevated levels of cortisol (Sulikowski et al. 2006), which suggests that stocking density can, indeed, act as an environmental stressor.

One method which may reduce aggressive behavior, even at high stocking densities is manipulating photoperiod. Studies with Japanese flounder have shown that fin nipping and other signs of aggressive behavior only occur during the day and not at night (Sakakura and Tsukamoto 2002). In the past, we have reared winter flounder using 24L:0D photoperiod to promote growth, however, in hindsight, this may have been deleterious to the fish. A constant light photoperiod regime may have increased aggressive behavior resulting in stressed and fin damaged fish.

Our working hypothesis is that juvenile winter flounder held at high stocking densities under constant light are physiologically stressed, which may be due to increased aggressive behavior. The aggressive behavior of fin nipping causes some initial damage to the fins, and this is exacerbated because the stress leads to a decreased resistance to bacterial infection. To test this hypothesis, we will conduct an experiment examining the effect of stocking density and photoperiod on the incidence of fin erosion.

The experiment follows the general experimental design and methodologies used by Fairchild and Howell (2001). A 2x3 randomized complete block experiment with 3 replicates was initiated on 26 June 2006 in which 57 dph flounder were stocked out into 2 photoperiod treatments (24L:0D, 12L:12D) and 3 density treatments (20, 100, 300%). Density is measured as the ratio of total fish area to tank bottom area. A total of 1,614 fish were used such that 13, 64, and 192 fish were stocked into each of the 20, 100, and 300 treatments, respectively. Initial aquaria consist of 3-L plastic gardening pots with a 182 cm² bottom surface area. The aquaria are set up in water tables and each is supplied with individual water lines connected to a flow-through system. Overhead lights on timers provide the photoperiod treatment. Stocking densities will be adjusted over time as the fish grow by moving the fish into progressively larger aquaria.

Fish are monitored daily which includes feeding (Gemma 0.3 mm), removing excess waste and any mortalities, and measuring water temperature, dissolved oxygen and salinity. Each week, 10 randomly selected fish from each replicate are removed. Fish are anesthetized (50 ppm MS-222), measured (TL) and weighed (wet weight). Each fish is examined under low magnification, and a qualitative score of fin erosion is assigned to. Scores range from 0 (complete fin erosion) to 5 (no fin erosion). Following data collection, fish are returned to their aquaria. In addition to fin erosion observations, the degree of aggressive behavior in each treatment is quantified at weekly intervals. In this, an observer spends 10 minutes observing the fish in each aquarium, and recording the number of fin-nipping incidents observed. Because we also are interested in the effects of stocking density on gender development, the fish from this experiment will be saved (in modified Davidson’s fixative) for later histological processing.

Northwest Fisheries Science Center Progress ­ January through June 2006
Select Appropriate Species:
In the first half of 2006, Washington SCORE scientists engaged with the Makah tribe over the tribes continually decreasing OY for Canary and Yelloweye Rockfish. SCORE scientists have agreed to help the tribe with design of a rockfish hatchery, and will reprogram future SCORE funds toward development of rockfish culture systems.

Develop a Genetic Management Plan:
Pacific cod genetic samples from 9 fish were collected during broodstock collection trips in Puget Sound. These samples were provided to scientists at NOAA’s Alaska Fisheries Science Center who are collecting samples from researchers throughout the north Pacific. This work will establish the genetic stock structure for this species and may indicated if Puget Sound stocks are unique.

Develop Culture Technology:
Given the reduced budget this year, SCORE Washington scientists had planned on concentrating on improving culture technology in two areas:
A) Scaling up Pacific Cod Hatchery techniques.
B) Determining optimal feeding timing in larval rockfish

Scaling up Pacific Cod hatchery techniques was only partially successful. While infrastructure was put in place to rear up to 10,000 cod juveniles for release, we were unsuccessful in obtaining a single live female cod during broodstock collection trips. This year only 9 adult cod were collected following 3 days of trawling, one week of long-lining and 4 weeks of hook and line fishing. Of the 9 fish only three were female and all three died shortly after collection. Of the remainder, 5 males are still alive and have been added to F1 fish from two years ago. The F1 fish may be of spawning age next year, so if we continue to be unable to collect wild Pacific cod, we will use our captive broodstock made up of F1 and wild (male) fish in 2007. This is the second year in a row that the broodstock did not show on the spawning grounds. Infrastructure developed for Pacific cod was tested with a group of Rock sole (lepidopsetta bilineata) and proved to be satisfactory.

The study to determine optimal feeding timing in larval rockfish will be started in July 2006.
Manage Disease and Health:
The pre-proposal for funding entitled “Proactive control of infectious diseases in emerging marine finfish aquaculture” developed by Mike Rust and Mike Kent of Oregon State University was not funded.

Describe Life History Patterns and Ecological Interactions and Optimize Release Strategies:

The pre- proposal for funding entitle “Demonstration of the effectiveness of conservation aquaculture for Puget sound Pacific Cod (Gadus macrocephalus)” developed by Barry Berejikian, Jonathan Lee and Michael Rust was not funded. However a scaled down version of the experiments developed as a part of the proposal will be applied to rockfish as a part of Dr. Jonathan Lee’s post-doc research.

Communicate Results and Network Stock Enhancement Researchers & Managers:
SCORE researchers have been meeting periodically to help plan and coordinate the 4-day long 3rd International Symposium on Stock Enhancement and Sea Ranching. SCORE scientists work closely with a marketing firm to promote the conference, helped this firm refine a web page to communicate about the conference, and serve on both the Steering Committee and the International Scientific Committee to help plan the conference program.