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 habitat attributes affect the movement 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. This year the broodstock spawned seven batches of eggs, but only three were of high enough quality to culture. Larvae were used in the greenwater and developmental morphology studies described below. Juvenile fish currently are being used for weaning experiments and will be used for field trials later this summer. One-year old juveniles from last year’s production run were released in the spring.

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). Flounder acclimation cages used at the release are a necessary tool that allows the stocked fish to adjust to their new environment (Fairchild and Howell 2004; Sulikowski et al. 2005, 2006), however they also can be detrimental by attracting predatory green crabs to the release site (Fairchild et al., in press). Modification of the release strategy is necessary to offset this problem and alternate release strategies are being investigated. For example, in August 2006, we tested the idea that fish could be acclimated in cages for the required 48 hr period, then gently towed to an alternate release site 250 m downriver, and immediately released to offset crab aggregations. However, to ensure that the move would not adversely affect the fish, indicated by an increase in cortisol levels, the following experiment was conducted. A total of 6 small acclimation cages (0.1 m²) were each stocked with 50 fish (118 dph; mean TL = 40 + 8 mm; 102% density) and secured to the bottom by divers on August 8 2006. Two days later, half of the cages were hauled to the surface and the fish were immediately snap frozen on dry ice. The other 3 cages were raised and tethered to the boat so that they were still submerged, and towed at < 1 kt to the alternate release site. There the cages were lowered to the bottom for 10 min to simulate release conditions, then raised, and all fish were snap frozen on dry ice. Samples for cortisol analysis were prepared according to Sulikowski et al. (2005).

Despite moving the fish in cages very gently to a nearby alternate release site, the fish experienced a significantly higher level of stress (ANOVA, df=1, p<0.001), as measured by cortisol concentration, than the caged fish which were not moved (Figure 1). This release strategy may be an effective way to bypass green crab congregations, but the surge in cortisol indicates that this method would be detrimental to the cultured fish.

Temporal and Spatial Distribution of Juvenile Winter Flounder in the Estuary:
The objective of this study is 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 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.

Wild juveniles:
Ten juvenile wild winter flounder (mean TL = 14.67 + 2.96 cm) were caught with a beam trawl in the Hampton River in September 2006 and tagged at sea. Tagged fish were then impounded in acclimation cages for 48 hrs to ensure that the tags were secure and fish were healthy. Fish were released from the cages and tracking was done using an array of 6 submersible receivers (VR2s) positioned in the Hampton River and at the mouth of the estuary. Eighty percent of the fish remained in the immediate release area for the first two weeks. As time at large increased, several fish dispersed downriver, and the largest individual (age 2) left the estuary two months after release in mid-December. A second fish (the third largest tagged) also left the estuary four months after release in mid-February. The remaining eight fish were tracked until the batteries in the tags expired (mid-March 2007). Final positions of the tagged fish indicated that 20% had left the estuary all together, 30% of the tagged fish were still at the release site, 20% were approximately 500 m downriver from the release site, and 30% were unaccounted for.

A final synopsis of the home ranges, dispersal patterns, and habitat associations of tagged cultured and wild juvenile winter flounder will be presented in October 2007 at the Second International Symposium on Tagging and Tracking Marine Fish with Electronic Devices in San Sebastián, Spain.

The Use of Concentrated Algae as a Replacement for Live Algae for Greenwater:
The addition of live microalgae into larval fish culture water to enhance fish growth and survival is standard practice in the aquaculture industry. Microalgae or “greenwater” provides an added nutritional supplement to larval fish, as well as to the cultured zooplankton fed to these fish. In addition, the green color change in the culture water provides a contrast which may aide these young, developing fish in locating their prey. For instance, higher survival was observed in summer flounder larvae with the addition of greenwater to culture water (Alves et al. 1999). However, growing and maintaining live cultures of microalgae is a costly and time consuming process. With the advances in algae processing, using concentrated solutions of microalgae for greenwater are now possible. If larval fish growth and survival are as high using these greenwater products as they are with normal live microalgae, the ramifications for the aquaculture industry would be significant.

To determine if concentrated algae products promote better growth and survival in larval fish than live algae, and thus would be a suitable replacement, the following experiment was conducted. A 2x3 randomized complete block design experiment was set up at the UNH’s CML. Two replicates of 3 greenwater treatments were tested. These greenwater treatments were: 1) control (no microalgae in culture water), 2) Nannochloropsis Instant Algae (liquid concentrate) from Reed Mariculture, Inc. (520 E. McGliney Lane, Campbell, CA 95008), and 3) live Nannochloropsis grown at the CML.

Experimental units were 145-liter fiberglass tanks connected to a flow-through, ultra-violet filtered, ambient temperature, sea water manifold. Flow rate was maintained at 10-11 ml/s. resulting in a complete turnover every 3.7 to 4 hours. Two hundred fifty newly-hatched cultured winter flounder larvae were stocked into each tank on 10 May 2007. Fish were monitored daily for three weeks which coincided with the normal duration of greenwater use for winter flounder. Daily protocol included measuring water temperature, dissolved oxygen, salinity, pH, and light in each tank. Fish were fed Selco enriched rotifers at a density of 4000 prey/liter three times (at 800, 1400, 2000) daily. Tanks which received live algae were “greened” immediately prior to the 800 and 2000 feedings by gently pouring in 6 liters of live Nannochloropsis that had been chilled to the temperature of the fish culture water. For the Instant Algae treatment, 1.6 mls of concentrated Nannochloropsis (cell size ~ 1-2 microns; ~ 68 billion cells/ml; dry weight = 18%) were diluted in 1 liter seawater, and then gently poured into each of the two replicate tanks immediately prior to all three of the rotifer feedings. Control tanks did not have any microalgae added.

Each week, a subsample of 20 fish/replicate was measured to assess growth and developmental performance. At the end of the three weeks, mean weekly growth rates between treatments were calculated and analyzed using analysis of variance with Bonferrroni posterior tests. Survival data were analyzed using chi square tests.

Because significant differences (p<0.05) existed between replicates each week, individual tanks were analyzed independently of each other. Fish in all replicates grew each week and there was a lot of similarity in these growth rates (Figure 2). After three weeks, fish from the Instant Algae 2 tank were significantly longer (p<0.05) than fish from all the other replicates.

High mortality occurred during the first three weeks of the experiment in all replicates. However, survival was 2 to 7 times higher in the Instant Algae treatment than in either the control or live algae treatments (Figure 3).

Replicate tanks were pooled when greenwater use ended (24 dph) and the fish continued to be reared according to standard protocols (see Fairchild et al. 2007) and monitored as they developed into juveniles. Once pigmentation development was complete (52 dph), the fish were measured, photographed, and indexed to determine if the type of greenwater treatment affected the rate of abnormal pigmentation in the cultured flounder. Finally, all fish were snap-frozen and stored in a -80°C freezer until fatty acid (DHA) profile analysis (which will assess the nutritional value of the greenwater treatments) is conducted.

Fish continued to grow in the subsequent four weeks (Figure 4). Fish in the Live Algae treatment consistently were the smallest and fish from the Instant Algae treatment were always the largest by week 6. At the end of the study, neither Control nor Live Algae treatments contained any fish with malpigmentation. In the Instant Algae treatment, 12.9% of the fish had malpigmentation; all of these individuals were devoid of any pigmentation on the ocular side.

Surprisingly, fish cultured in live algae during the three weeks of greenwater use usually showed slower growth than either the fish cultured without algae or cultured in Instant Algae. It could be that the live algae was not exceptionally concentrated during portions of the experiment or had some undetected pathogen that was harmful to the larvae. Had the live algae cultures been better, presumably these fish would have had better growth and survival rates. However, rearing live food, whether zooplankton or microalgae, for fish culture is subject to failure; Instant Algae eliminates difficulties with the microalgae cultures. Larvae cultured in Instant Algae exhibited good if not better growth rates than the other treatments, plus had superior survival. Culture water was greened three times daily in the Instant Algae treatment in contrast to only twice daily in the live algae treatment. The rationale for this was if a facility were to use concentrated algae, it would be readily available for all feedings, whereas live algae production can be limited. In past years, we have only been able to produce enough live algae at the CML for two feedings per day so it has become the standard for our winter flounder rearing protocol.

One drawback was that only fish cultured in Instant Algae had pigmentation problems. However, the proportion of abnormally pigmented fish was relatively low compared to the higher survival of fish in this treatment. For example, though all fish would be normally pigmented, only 6 and 5 fish out of 100 would survive in the Control and Live Algae treatments, respectively. In the Instant Algae treatment, 36 out of 100 fish would survive, with only 5 fish abnormally pigmented. Given these circumstances, we recommend using Instant Algae for greenwater culture of larval winter flounder.

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 two studies were initiated 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 are being 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 are now being sampled once a week from 40 DPH to 80 DPH. For histological examination of the gut, five specimens are 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 are 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 are being 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 are fed three times/day to satiation (approximately 10-15% of body weight/day). Water temperature, salinity and dissolved oxygen are monitored daily. In addition, in each tub excess food is removed, and 30-50% of the water is siphoned out and replaced each day. Weaning success will be measured throughout the five week experiment by examining specific growth rates and survival. A subsample of three fish/tank will be measured and weighed weekly. Mortalities will be removed daily. 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.

Outreach:
To test new elastomer tagging formulas and to educate the press on our stock enhancement program, a tag and release demonstration was executed this spring. On 6 April 2007, 1018 fish (mean length = 9.53 + 0.40 cm TL) were tagged with red VIE tags (new 1:1 formula) using the air injection tagging system with the aid of Geraldine Vander Haegen from Northwest Marine Technology. Tagged fish were transferred into cages within tanks which containing 2-4 cm sand. Fish were kept in these cages at a 458% stocking density until the release date so that they could adjust their color to the sand color and hone burial skills. While in cages, fish were fed once daily Gemma 1.2 diet.

On 11 April 2007, 998 366 dph fish were released into the Hampton River at low tide. (20 fish were retained in the CML for tag retention and mortality analyses) Fish were emptied from the cages into live wells on trucks and transported to the release site (approx. 45 min transport time). Fish density was 683% in the live wells. Once at the release area, fish were dip netted into 5-gallon buckets, carried down to the shore, and gently released from the buckets in approximately 0.5 m deep water at low tide. Each bucket of fish was emptied adjacent to the previous bucket moving up river so that fish density was uniform and sparse. Reporters and photographers from UNH media relations, The Union Leader, The Portsmouth Herald, and New Hampshire Public Radio were on hand to document the release. Participants included all UNH SCORE personnel plus several graduate students.

In June approximately 500 60 dph juvenile fish were given to MIT Sea Grant for their Aquaculture in the Classroom Program. These fish will be used in a variety of aquaculture-related experiments in grade schools throughout Massachusetts in the 2007-2008 academic year.

Presentations:
Fairchild, E.A., W.H. Howell, and N. Rennels. 2007. A decade of research: current status of the winter flounder Pseudopleuronectes americanus stock enhancement program. Aquaculture 2007. The annual meeting of the World Aquaculture Society, February 26 - March 2, 2007, San Antonio, TX. (session co-organizer and moderator).

Publications:
Fairchild, E.A., N. Rennels and W.H. Howell (In Press). Effectiveness of acclimation cages for winter flounder stock enhancement. Reviews in Fisheries Science.

Fairchild, E.A., N. Rennels, W.H. Howell and R.E. Wells. 2007. Gonadal development and differentiation in cultured juvenile winter flounder, Pseudopleuronectes americanus. Journal of the World Aquaculture Society 38(1): 114-121.