Science Consortium for Ocean Replenishment and Enhancement 2004 Annual Report

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

Because of size at release is an important variable, and because we have released relatively small fish (5-6cm) in the past, we chose to hold some of the 2003 fish in the laboratory over the winter until they reached a larger size. In late June 2004, these cultured fish (n=562, mean size=10.9 cm, age=14 months) were tagged with a mixture of tags including, Visible Implant Elastomer Tags, Decimal Coded Wire Tags‘, and Visible Implant Alphanumeric Tags, and released in the Hampton-Seabrook estuary. The release was 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. Weekly sampling for these fish, at the 5 sampling stations, continued through October 2004. None of these fish were recaptured.

In addition to the fish described above, in the spring of 2004 we produced approximately 2,000 winter flounder juveniles for a second 2004 releases. These fish (mean size=5.9 cm, age=6 months) were tagged in September with Decimal Coded Wire Tags‘, acclimated as decribed above, and released into the Hampton-Seabrook Estuary in late September. In addition to these cultured fish, 64 wild juvenile winter flounder were caught at the release site in the preceding days, tagged with Visible Implant Alphanumeric Tags, and released. Because of our lack of success in recapturing fish from the earlier release, we changed our sampling protocol. We hypothesized, based upon recent results from a European study with a similar species, that our fish were not dispersing away from the release location, and that our sampling program, with five sampling locations spread over about 15 km2, was too large. Thus we established a grid 300m long by 100m wide (3,000 m2), divided into 50m intervals in both directions. The release location was at the center of this grid. Observations of the released fish began immediately. This was facilitated by 15 of the fish being tagged with large, red streamer tags that were clearly visable to the divers who released the fish from the acclimation cage. They observed that the released fish moved only a few meters from the release site. Sampling for released fish began the day of release, and continued over the next seven days. Replicate beam trawls were made at every intersection of the grid (21 stations). On the day of release, beam trawl tows were made at each grid intersection every 3 hours, beginning one hour after release. Each grid intersection was repeatedly sampled over the following 6 days. Fourteen cultured winter flounder were caught through this effort. Of noteable importance, all cultured fish were recaptured within meters of their release location while wild juveniles were recaptured at all locations within the 3000m2 sampling grid. This confirmed our hypothesis that the released, cultured fish do not quickly disperse away from the release site, and indicated that the scale at which we has been sampling in the past was probably too large. The low numbers recaptured may indicate that mortality of released fish is higher than we previously believed. Because the divers observed a large aggregation of green crabs around, and presumably attracted by, the acclimation cages, we believe the source of mortality may be green crab predation. To alleviate this, we will move the acclimation cages away from any crab aggregation in all subsequent releases. Gut contents of a sample of wild juveniles were compared to the gut contents of all recaptured cultured fish. Results indicated that the wild juveniles had more in their stomachs, and had a more diverse diet than cultured fish. One of the greatest differences was that the cultured fish contained small mussels, which, because of their size and color, resemble the formulated diets the cultured fish had been, provide. This suggests that cultured fish differ from wild fish in their feeding ecology, at least in the short term, and that this difference may be due to their feeding history.

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 have established a sampling regime to determine the seasonality of: 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 to record temperature and salinity at hourly intervals. Surveys of these stations began in June and continued through October 2004. In addition to these abiotic measurements, 3 types of collecting gear were used at each station on each weekly sampling occasion. In shallow water (<1.5m) we employed a 17m x 2m beach seine. Three replicate samples, each with an approximate swept area of 550m², were taken near low tide. In mid-depth areas (1.5-3m) we employed a 1m beam trawl. Three replicate 50m long tows were taken within the 1.5-3m depth interval, each parallel to the shore. In the deeper areas (>3m) we used 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 were taken within the depth interval, each parallel to the shore, and each approximately 100m length. The catch from all sampling has been identified, enumerated, and measured. All winter flounder caught were checked for tags. Abundance was 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 took a bi-weekly series of 6 replicate benthic cores (0.0079m² to a depth of 10cm) at each station. Cores were stored in Zip-Lock™ bags, placed in ice, and returned to the laboratory where they were sieved through a 1mm mesh sieve. All prey taxa are stained with Rose Bengal and preserved in 10% buffered formalin. All will be identified, counted, and weighed in the next several weeks.

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 have been studying sexual differentiation and sex ratio of cultured fish as part of this study. Starting at metamorphosis, a total of 376 cultured fish were sampled from the general hatchery population at approximately 10 mm total length (TL) intervals at 110, 124, 160, 231, and 323 days post-hatch. On each sampling occasion, randomly collected fish were euthanized by an overdose of anesthesia (MS-222), measured, and weighed. Fish were fixed and stored in modified Davidson’s fixative for at least 48 h. Prior to histological processing, the samples were washed in freshwater and stored in 70% alcohol. Histological processing involved embedding the fish in paraffin, sagittal sectioning (5 _m), and staining with hematoxylin and eosin. Slides were numerically coded and examined by three viewers in a blind test to determine fish sex based on structures and cells associated with gonadal tissue. Fish were scored male, female, unknown (gonadal tissue visible but unidentifiable to sex), or missed (no gonadal tissue present on slide). Sex ratio data were calculated and analyzed using Chi-square goodness of fit. Those fish that were scored as “unknowns” or “missed” were excluded from the statistical analyses. In addition, to determine if fish age or size affected the sex ratio, the data were sorted accordingly and reanalyzed using Chi-square goodness of fit tests. Results suggest that sexual differentiation in winter flounder occurs at a size < 41 mm TL. A total of 65 females and 162 males were identified from the 41-110 mm TL cultured flounder population yielding a sex ratio that was significantly skewed towards male (_2=44.7, df=1, p<0.001). This trend held true when cultured fish were sorted by age and size, with the exception of those fish 61-70 mm TL; an aberration probably due to a small sample size.

Stress Physiology

Because hatchery reared fish are handled as they are tagged and transported to the release site, it is important to understand how these practices stress the fish, and what impact this has, if any, on their survival and performance after release. 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.

Publications:
Fairchild, E.A. and W.H. Howell. 2004. Factors affecting the post-release survival of juvenile Pseudopleuronectes americanus. J. Fish Biol. 65: 69-87.

Sulikowski, J.A., E.A. Fairchild, N. Rennels and W.H. Howell. (In Press). The effects of tagging and transport stress on juvenile winter flounder, Pseudopleuronectes americanus: implications for successful stock enhancement. J. World. Aqua. Soc.

Fairchild, E.A., J. Fleck, and W. H. Howell (In Review). Determining an optimal release site for juvenile Pseudopleuronectes americanus in the Great Bay Estuary, New Hampshire, USA. J. Aquaculture Research.

Sulikowski, J.A., E.A. Fairchild, N. Rennels and W.H. Howell. (In Review). The effects of transport density on stress in juvenile winter flounder, Pseudopleuronectes americanus: implications for successful stock enhancement. J. World Aqua. Soc.

Fairchild, E.A., N. Rennels and W. H. Howell. (In Prep.for J. World Aqua. Soc.). The winter flounder Pseudopleuronectes americanus gender bender: what are we culturing?

Professional Presentations:
Fairchild, E.A., J. Sulikowski, W.H. Howell and N. Rennels. Pilot scale release of cultured, juvenile winter flounder, Pseudopleuronectes americanus, into the Hampton-Seabrook Estuary, New Hampshire,USA. World Aquaculture Society Annual Meeting, Honolulu, HI, March 2004.

Fairchild, E.A. and W.H. Howell. Winter flounder stock enhancement: deficits in cultured fish. International Symposium on Nature and Culture: Comparative Biology and Interactions of Wild and Farmed Fish, Imperial College, London, July 2004.

Fairchild, E.A., W.H. Howell. N. Rennels and J. Sulikowski. The winter flounder gender bender: what are we culturing? Ninth Annual Flatfish Biology Conference, Westbrook, CT, December 2004.