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, and to understand how developmental morphology and live feed conditioning affects post-release survival of juvenile winter flounder. Elements of the program addressed in this reporting period have included:

Fish Production:
The wild-caught winter flounder broodstock was brought into the Coastal Marine Laboratory (CML) in March. Larvae were used in developmental morphology studies. Juvenile fish were used for weaning experiments and field trials in the summer and fall. Several hundred juveniles are being maintained for spring studies.

Temporal and Spatial Distribution of Juvenile Winter Flounder in the Estuary:
The objective of this study was to identify the areas within the estuary where juvenile fish are found and when, to characterize their habitat, and to study their temporal and spatial use of the estuary. To accomplish this goal, juvenile winter flounder were anesthetized, fitted with acoustic tags (VEMCO V7-2L-R256 coded pinger tags), and released. Each acoustic transmitter emitted a distinctive coded pulse (frequency 69 khz) that was detected by a hydrophone, thereby allowing the fish’s location to be accurately determined, and the fish’s movements to be tracked over time.

Sediment analyses where cultured and wild age1 fish were located were completed during this reporting period. Sediment grain size composition analyses were processed according to protocol developed by Folk (1980). The mean percent gravel, sand, clay, and silt of the sediments at the location where fish were released and the locations where cultured and wild fish were found are reported in Table 1.

Percent sediment composition data were arcsine transformed (Zar 1996). Differences in particle sizes between the sites where cultured and wild fish were found were tested with analyses of variance. No significant (p>0.05) differences were found between any of the grain size fractions. These results indicate that released cultured winter flounder will inhabit the same sediment types as similarly-sized wild fish.

A final synopsis of the home ranges, dispersal patterns, and habitat associations of tagged cultured and wild juvenile winter flounder was presented in October 2007 at the Second International Symposium on Tagging and Tracking Marine Fish with Electronic Devices in San Sebastián, Spain. We plan to publish this study in the conference proceedings.

Developmental Morphology and Weaning Studies:
A major challenge of any captive rearing program, whether for aquaculture or stock enhancement, is providing the appropriate diet regimes during development. Typically marine fish larvae are initially fed live food (e.g. rotifers, Artemia), and are then weaned onto formulated diets as they attain a size or developmental state that supports consumption of formulated diets. Weaning onto formulated diets is a stressful time for cultured fish, and this is especially true for flatfish that are concurrently undergoing dramatic morphological and physiological transformations associated with metamorphosis. Weaning occurs twice for fish that are used for stock enhancement; the second time occurs as they transition from formulated hatchery feed back onto live diets after release. To successfully wean fish for stock enhancement during these sensitive early life stages, we need to fully understand the ontogenetic development of the digestive system. We also need to identify diets that will optimize weaning success in the laboratory and minimize the effects of subsequent weaning in the wild. Finally, we need to examine the transition onto natural diets once reared individuals are released and investigate dietary differences between recently released and wild stocks. To begin to address these areas, the following studies were continued during this period.

Describing the development of the digestive tract of winter flounder:
The morphological changes of the digestive system and its associated structures from hatching through the post-metamorphic juvenile period are being described. Fish were sampled on a schedule covering the entire early life-history period. Individuals were sampled daily from 0 (hatching) to 20 DPH, three times per week from 20 to 40 DPH, and sampled once a week from 40 DPH to 80 DPH. For histological examination of the gut, five specimens were fixed in modified Davidson’s solution on each sampling occasion. Gut epithelial cell development (stomach and intestine) will be observed using light microscopy at various magnifications. An additional five specimens on each sampling occasion were preserved in 10% buffered formalin for examination of the morphometric changes in the digestive tract. The digestive organs will be digitally photographed under a microscope for relative size and orientation measurements (esophageal, stomach, and intestinal length, shape and area).

Examining weaning success of juvenile winter flounder:
We will determine how live diet regimes affect the success of weaning as indicated by growth rate and survival in juvenile winter flounder. Ninety, newly-settled, 55 dph juveniles were distributed on 28 June 2007 into each of nine 20-l flow through, circular tubs (46 cm dia. x 32 cm deep; 10 individuals per tub) and fed one of three different weaning diets (three replicates per weaning diet):
1) formulated commercially available microparticulate diet (Skretting “Gemma”)
2) live white worms (Enchytraeus albidus)
3) control (no weaning - continued Artemia).

Fish were fed three times daily to satiation (approximately 10-15% of body weight/day). Water temperature, salinity and dissolved oxygen were monitored daily. In addition, in each tub, excess food was removed, and 30-50% of the water was siphoned out and replaced each day. A subsample of three fish/tank was measured and weighed weekly. Mortalities were removed daily. Weaning success will be measured throughout the five week experiment by examining specific growth rates and survival. Differences among treatments in specific growth and survival among the three weaning diets will be assessed with MANOVA followed by individual one-way ANOVAs (repeated measures design) and a Tukey's Post Hoc Test. In addition, a subsample of fish from each experimental unit (tub) and a sample of similarly-sized wild-caught fish from New Castle, NH were snap-frozen on dry ice and sent to the NMFS Milford Laboratory for RNA/DNA analyses as part of a collaborative research project.

A second similar, but preliminary, experiment was conducted during this reporting period. In it, fish were fed marine amphipods or grindal worms. The experiment will be analyzed and likely repeated in 2008.

Examining onset of wild weaning in pellet-reared fish
We will determine the onset of weaning onto natural diets in the wild as indicated by the presence and type of food in the gut and by gut fullness. Pellet-reared fish (45-65mm TL) were released in 90cm x 60cm x 45cm cages (N=10 fish per cage) in a mud-silt bottomed, eelgrass cove of the Piscataqua River Mouth, New Castle, New Hampshire. Fish were starved 24 hours prior to release to allow evacuation of the gut and to invoke hunger. Preliminary trials indicated that fish began feeding in the wild within 48 hours of release; therefore, cages were retrieved every 3 hours up to 51 hours post release (=17 cage retrievals). Fish were immediately frozen for later gut analyses. Wild fish of similar size were collected by seine net near the release site for dietary comparison. This protocol was repeated in the Hampton-Seabrook estuary, 17 miles south of the initial release site where wild fish are more abundant; however, sediments are courser (silt-sand) and include scattered mussel beds.

Creating a Specific Diet for Winter Flounder:
No species-specific diet exists for winter flounder; we use a standard coldwater marine finfish diet produced by Skretting. In August, we began a cooperative venture with Cargill to formulate and manufacture a US made diet specific to winter flounder. Cargill’s beta test diet did not perform as well as the Skretting diet; fish fed Skretting diet grew larger and faster than fish fed the beta test diet. Texture was a noticeable difference between the diets with the Cargill diet being rougher than the Skretting diet. We anticipate repeating this feeding trial in summer 2008 with a different formulated Cargill beta test diet.

Outreach:
In November, juvenile winter flounder were donated to the Seacoast Science Center in Rye, NH, an educational organization that focuses on coastal environmental history and exhibits. The flounder are being used in a “creature feature” display on indigenous fishes of the Gulf of Maine. Information about on-going winter flounder stock enhancement research at UNH is provided at the exhibit.

In December, approximately 200 juvenile fish were given to Rich Bell, a Mater’s student at the University of Rhode Island. These fish will be used in a variety of aquaculture-related experiments.

Presentations:
Fairchild, E. A., N. Rennels, and W. H. Howell. 2007. Movements and habitat use of cultured and wild juvenile winter flounder Pseudopleuronectes americanus in a shallow estuary. Second International Symposium on Tagging and Tracking Marine Fish with Electronic Devices. October 8-11, 2007, San Sebastian, Spain.

Walsh, M. L. and W. H. Howell. 2007. Winter flounder movements off southern New Hampshire. Second International Symposium on Tagging and Tracking Marine Fish with Electronic Devices. October 8-11, 2007, San Sebastian, Spain.

Publications:
Fairchild, E. A., J. A. Sulikowski, N. Rennels, W. H. Howell, and C. W. D. Gurshin. (In Review). Distribution of winter flounder, Pseudopleuronectes americanus, in the Hampton-Seabrook Estuary, New Hampshire: observations from a summer field study.