Comparison of stress responses in wild and captive winter flounder (Pseudopleuronectes americanus Walbaum) broodstock

Chronic stress is responsible for many problems occurring in fish holding facilities, such as increased susceptibility to disease, reduced growth rate and suppression of the immune response. The goal of this study was to verify if wild winter flounder (Pseudopleuronectes americanus Walbaum) suffer from chronic stress when kept several months in captivity. We hypothesized that winter flounder not subjected to chronic stress in captivity would have similar or higher condition indices and similar stress response compared to fish from the wild. Our results showed that the condition index of winter flounder kept in captivity was higher (1.50) than those from the wild (1.33) even after one year of captivity. The intensity of the acute stress response following short‐term exposure to air was similar between wild and captive fish (no significant interaction between the factors ‘stress’ and ‘origin of the fish’). Body water content (an indicator of energy reserves) was lower (76.1%) compared to wild fish (79.8%) after one year of captivity, suggesting that the winter fast had been less energetically demanding in captive fish. These results indicate that no chronic stress was present in captive fish. On the other hand, the mortality during captivity was around 30%, which is high for the aquaculture industry. Further work to address this problem is required.     

Determining an optimal release site for juvenile winter flounder Pseudopleuronectes americanus (Walbaum) in the Great Bay Estuary, NH, USA

One of the main elements in developing an optimal release strategy for an enhancement effort is to evaluate and select release sites that will support growth and survival of newly released, cultured fish. Three potential release sites (New Castle (NC), Broad Cove (BC) and Oyster River (OR)) in the Great Bay Estuary, NH, USA were evaluated for pilot‐scale releases of winter flounder (Pseudopleuronectes americanus Walbaum). Cultured juvenile flounder were placed in cages at each of the three sites. Sites were evaluated based on growth and survival of the fish in relation to water temperature, prey availability and sediment composition. Fish grew faster in the two upper estuarine sites BC (0.54 mm day^?1) and OR (0.56 mm day^?1) than at the site at the mouth of the estuary (NC=0.37 mm day^?1). Fish survival (44–53%) and water temperature (17.8–19.7°C) did not vary between sites. Benthic samples showed that prey was available to, and eaten by, the flounder. Sediment composition was the main difference between the three sites, with one site (NC) characterized by gravel whereas the other two sites were sandy. These results corroborate other studies showing the importance of sediment quality for the distribution of flatfish populations. From these results, we can confidently eliminate NC as a potential release area and recommend that sandy sites are better for stocking cultured juvenile winter flounder than gravely sites.     

Influence of gonadotropic hormone-releasing hormone analog (GnRH-A) on plasma sex steroid profiles and milt production in male winter flounder,Pseudopleuronectes americanus (Walbaum)

The effects of gonadotropic hormone-releasing hormone analog (GnRH-A) treatment on the onset and duration of increases in plasma sex steroids and milt production (milt volume and number of spermatozoa) were investigated in prespawning male winter flounder. After treatment of maturing males during the winter with a single injection of either 20 or 200 g/kg [D-Ala⁶, Pro⁹-NHEt]LHRH (GnRH-A), plasma levels of testosterone and 11-ketotestosterone were increased within 12h and the steroid hormone levels remained elevated for long periods lasting several days. The androgenic steroid response of males was delayed after the administration of a lower dose of GnRH-A (2 g/kg). Although a single GnRH-A injection in December or January advanced the onset of spermiation in some males, only small amounts (<50 l) of milt could be collected. By March, all males were in spermiating condition following GnRH-A treatment; however, significant increases in sperm production, particularly increases in milt volume, occurred in fish twice treated with GnRH-A.Part of this work presented at the First Symposium of Fish Endocrinology, Edmonton, Alberta, Canada June 1988 and the Satellite Symposium on Applications of Comparative Endocrinology to Fish Culture, Almuñecar, Granada, Spain May 1989     

Influence of temperature on growth and survival of juvenile winter flounder,Pseudopleuronectes americanus (Walbaum) reared under continuous light

Winter flounder, Pseudopleuronectes americanus, has emerged as a promising candidate flatfish for cold‐water aquaculture and restocking. Here, juveniles were reared for 8 weeks at three temperatures: 10, 15, and 20°C under 24‐hr light. All fish were imaged at stocking and at 2‐week intervals, where growth was measured as changes in standard length (S_L) and body area (B_A). By week 2, fish reared at 15 and 20°C were larger than those grown at 10°C. At weeks 4 to 6, fish at 15°C were larger than fish at 20°C. Linear regressions were used to model growth dynamics over time at each temperature. Highly significant linear growth trajectories were detected over time for S_L and B_A. S_L and B_A regressions also showed a significant difference among the slopes across temperatures, where comparing slopes showed the best temperature to rear the flounder was 15°C. Weights of fish held at 15°C and 20°C were greater than at 10°C at the termination of the experiment. Within each temperature, the growth rate of malpigmented fish was not different from that of the normally pigmented fish. Overall, growth of winter flounder was comparable to that of other commercially produced flatfish species, providing strong evidence for this flatfish species as a potential species for aquaculture.     

Bacterial colonization of winter flounder Pseudopleuronectes americanus fed live feed enriched with three different commercial diets

The proliferation of bacteria in intensive aquaculture systems may be responsible for poor growth and mass mortality of marine fish larvae. Essential fatty acids provided in the diet could protect larvae by modulation of the immune response via arachidonic acid (AA) and eicosapentaenoic acid (EPA). Winter flounder Pseudopleuronectes americanus larvae were fed rotifers Brachionus plicatilis enriched with three commercial diets containing different fatty acid profiles. Bacterial colonization on the gills and skin and in the intestinal lumen was evaluated at the end of the rotifer feeding period (day 26), and growth was surveyed until metamorphosis. At 26 days post hatching, larvae fed rotifers containing the higher AA content and with a higher docosahexaenoic acid (DHA) to EPA ratio showed better growth and the lowest bacterial colonization of the intestinal lumen compared to larvae fed rotifers with the lowest AA and DHA : EPA levels. AA had been selectively incorporated into the polar lipids of larvae fed the rotifers enriched with the three diets. This is the first study in winter flounder larvae to report a link between different commercial rotifer enrichments and bacterial density in intestinal lumen.     

Growth and survival of winter flounder (Pseudopleuronectes americanus) larvae reared on different photoperiod regimes from hatch to metamorphosis

The aim of this study was to determine the effect of photoperiod on growth and survival of winter flounder, Pseudopleuronectes americanus, from hatch until metamorphosis. Sixteen 100 L upwelling tanks were stocked with larvae at an initial stocking density of 20 larvae/L. Larvae in eight tanks were provided with continuous light (24 hr light/0 hr dark), while those in the remaining eight tanks were grown under a photoperiod of 14 hr light/10 hr dark. Tanks were sampled for larval standard lengh from 6 to 28 days post‐hatch, and the number of live larvae, live metamorphosed and dead metamorphosed fish were recorded at the termination of the experiment (starting 46 dph). Results indicated that larvae provided with continuous light were larger than those sampled from 14 hr light/10 hr dark treatment. No effect of photoperiod on the number of non‐metamorphosed fish recovered at the end of the experiment was detected. On the contrary, over three times as many fish survived and reached metamorphosis under continuous light. An ANCOVA model showed a significant interaction between light and age. Both regressions were significant and provided the following growth equations: SL_{24 hr light/0 hr dark} = 0.101 × age + 2.55 (r² = 0.96) and SL_{14 hr light/10 hr dark} = 0.094 × age +2.45 (r² = 0.97). Using 6.36 ± 0.059 mm SL as the mean size at metamorphosis, larvae experiencing continuous and 14 hr light/10 hr dark photoperiods would reach metamorphosis in 38 and 42 days respectively.     

Early weaning of winter flounder (Pseudopleuronectes americanus Walbaum) larvae on a commercial microencapsulated diet

Like most small marine fish larvae, the stomachs of winter flounder Pseudopleuronectes americanus are undeveloped at first feeding and have relatively reduced digestive capacity. This work was undertaken to test whether larvae at the onset of stomach differentiation (larval size about 5.5 mm) could be early weaned onto a commercial microencapsulated diet. We assessed the effect of early weaning by first comparing growth performance (standard length, total protein content and age at metamorphosis) of larvae fed enriched live prey from first feeding to a size of 5.5 mm and then reared on three different feeding regimes until metamorphosis: (1) live prey (LP) as a control group; (2) mixed feeding of live prey and microencapsulated diet (LP‐ME); (3) exclusively microencapsulated diet (ME) after fast weaning over 4 days (to a larval size of 6.2 mm). No differences were observed between larval development in the two first groups, which began metamorphosis at 40 days old. The larvae of the third group showed significantly slower growth that resulted in a delay of 4 days in the onset of metamorphosis. Differences in live prey availability between the treatments and the short transition period to allow the larvae to adapt to the new diet were identified as possible contributing factors to the slower growth and to the delay in metamorphosis of early weaned larvae. In a second experiment, the transitional weaning period was increased until the larvae were 6.6 mm in length. Weaning at that size resulted in no slowing of growth or delay in metamorphosis, suggesting that the feeding schedule was adequate.     

The development of a sperm cryopreservation protocol for winter flounder Pseudopleuronectes americanus (Walbaum): evaluation of cryoprotectants and diluents

Three cryoprotectants (dimethyl sulphoxide, propylene glycol and glycerol) and two diluents (sucrose based and saline based) were mixed (9 parts diluent–1 part cryoprotectant) factorially to produce six extenders that were tested to develop an effective sperm cryopreservation protocol for winter flounder Pseudopleuronectes americanus (Walbaum). Sperm were diluted 1:3 with each extender and frozen by flotation on liquid nitrogen before being submerged and stored for 30 days. Sperm left unfrozen in each extender for 20 min showed no toxic effects on motility. Extenders containing propylene glycol (PG) as cryoprotectant yielded higher post‐thaw sperm motilities than those containing dimethyl sulphoxide (DMSO) or glycerol. The sucrose‐based diluent performed better than the saline‐based diluent when DMSO was used as cryoprotectant, but there were no differences in post‐thaw motility between diluents for the other cryoprotectants. Activating sperm with ovarian fluid and sea water instead of sea water alone had no effect on post‐thaw motility. In fertilization trials, no differences were observed between any of the extenders and fresh milt when milt, eggs and sea water were left in contact for 1 h. When sperm were forced to compete for eggs by reducing contact time to 20 s, fertilization results followed those of sperm motility rates. Percentage hatch and morphology of larvae at hatching did not differ for eggs fertilized by cryopreserved and fresh sperm. This study represents the first reported successful attempt at cryopreserving winter flounder sperm and should improve gamete and broodstock management protocols for this species.     

Evidence for the presence of a viral non-lymphocystis type disease in winter flounder, Pseudopleuronectes americanus (Walbaum), from the north-west Atlantic

Abstract. A non-lymphocystis type disease associated with virus particles has been documented in winter flounder, Pseudopleuronectes americanus (Walbaum), taken from a small estuary in Conception Bay, Newfoundland. The papillomatosis-type lesions found on the dorsal surface of the fish contained numerous electron-dense particles, approximately 30 nm in diameter. A number of lines of evidence, including cytochemical studies, indicated that the particles were not glycogen but virions. Sensitivity to deoxyribonuclease enzyme treatment and the presence of crystalline viral'factories' in the nuclei of infected cells further suggested that the particles were small DNA virions. Characteristics including nucleic acid composition, particle size, site of multiplication and the lack of an envelope indicate that this fish virus is similar to the Papovaviridae and Parvoviridae families of mammalian viruses. Lymphocystis is common in flatfish populations along the Newfoundland and Labrador coasts of the N.W. Atlantic, but this is the first record of flatfish infection with a small DNA virus.     

Feeding larvae of winter flounder Pseudopleuronectes americanus (Walbaum) with live prey or microencapsulated diet: linear growth and protein, RNA and DNA content

A commercial microencapsulated diet was used as a total or partial replacement of live prey for feeding larvae of winter flounder Pseudopleuronectes americanus (Walbaum), a potential alternative finfish species for coldwater marine aquaculture. Growth performance (morphometric measurements and biochemical composition) and nutritional condition (RNA/DNA ratios) of larvae fed live prey (Brachionus plicatilis Müller), a microencapsulated diet or a mixed diet of live prey and microcapsules were compared. Newly hatched larvae were unable to digest microencapsulated diet; live prey at initial feeding was required for their survival and growth. Larvae offered a mixed diet showed slower growth than larvae fed exclusively with live prey. However, at the onset of stomach differentiation, RNA/DNA ratios (indicators of protein synthesis potential) of the larvae fed both diets became similar. We suggest that, at that stage (size 5.5–6.3 mm), enzymatic activity had developed enough to allow digestion of inert food. As the RNA/DNA ratio is a good indicator of nutritional condition, it appears to be an interesting tool for the assessment of diet adequacy in marine larval feeding technology.     

Trichodina murmanica (Ciliophora) and Gyrodactylus pleuronecti (Monogenea) parasitizing hatchery-reared winter flounder, Pseudopleuronectes americanus (Walbaum): effects on host growth and assessment of parasite interaction

Winter flounder, Pseudopleuronectes americanus, caught in Passamaquoddy Bay, New Brunswick, Canada, and held captive under crowded conditions, developed mixed infections of Trichodina murmanica Polyanski, 1955 (Ciliophora) and Gyrodactylus pleuronecti Cone, 1981 (Monogenea). A protocol involving sequential sieving was used to separate the two species of parasites and produce viable experimental baths. Replicate groups of juvenile, hatchery-reared flounder received one of the following treatments: mixed bath of G. pleuronecti and T. murmanica , bath of G. pleuronecti, bath of T. murmanica or parasite-free (controls). The abundance of both parasites correlated negatively with condition factor of the flounder ( r =−0.354, P  < 0.001 for Trichodina ; r =–0.205, P  < 0.05 for Gyrodactylus ). During the periods of peak parasite abundance (1–2 weeks postinfection), the effect of the two parasite species was additive, as mean condition factor and the percentage change in weight were significantly lower (ANOVA, P  < 0.05) among fish with mixed infections compared to single infections or controls. The most common signs of tissue pathology were increased density of epidermal mucous cells on the fins and macrovesicular lipidosis of the hepatocytes. After a significant decline in parasite infrapopulations (3 weeks postinfection), infected fish resumed normal growth, indicating the observed effects were somewhat reversible.     

Survival of larval summer flounder Paralichthys dentatus on formulated diets and failure of thyroid hormone treatment to improve performance

Summer flounder is a new aquaculture species, the culture of which could be made simpler by the replacement, to the greatest extent possible, of live feed with formulated diets. Previous work has shown that development of the summer flounder stomach can be accelerated by treatment of the larvae with thyroid hormone. Two series of experiments were conducted. In the first experiment, 2-week feeding trials were conducted beginning with fish (which were not treated with thyroid hormone) 14, 21, 28, 35, or 42 days after hatch (DAH) to determine when they could begin to utilize commercially available formulated diets. In the second experiment, two groups of sibling larvae, one treated with thyroid hormone, the other not, were placed in similar 2-week feeding trials at 25, 30 or 35 DAH to test whether survival of thyroid hormone-treated larvae would surpass those of untreated larvae. The first series of experiments demonstrated that the larvae could not effectively utilize the formulated diets until 35 DAH. The second series of experiments indicated that thyroid hormone-treated larvae do not exhibit significantly greater survival than untreated larvae.     

Monoaminergic substances in the teleost brain: Catecholamine levels in male and female winter flounder,Pseudopleuronectes americanus Walbaum,associated with gonadal recrudescence

High performance liquid chromatography with electrochemical detection (HPLC-EC) was used to quantitate catecholamine (CA) levels in the winter flounder brain following perchloric acid extraction/alumina purification of CNS tissues. Greater concentrations of norepinephrine (NE) and dopamine (DA) were present in the hypothalamus compared with the CA levels in whole brain. A seasonal study of CA brain levels in reproductively active male and female flounder demonstrated that monoamine levels reach their maxima in October in association with the rapid increases in gonadosomatic index. When perchloric acid extracts of the teleost and rat hypothalamus were submitted to direct HPLC-EC analysis, without alumina purification of CA neurotransmitters, similar hypothalamic profiles were obtained indicating the presence of identifiable biogenic amine neurotransmitters substances including NE, DA and serotonin (5-HT).     

Optimal dietary taurine level for growth of juvenile Japanese flounder Paralichthys olivaceus

The requirement for taurine in juvenile Japanese flounder Paralichthys olivaceus was determined by feeding diets containing various levels of taurine and cystine. Test diets supplemented with 0.5, 1.0 and 1.5% of taurine or with 0.5, 1.0 and 1.5% of L -cystine were prepared. The basal diet contained 55% protein from white fish meal. These diets were fed to juvenile Japanese flounder with an initial mean bodyweight of 0.9 g (total length (TL) 48 mm) for 5 weeks. Approximately 1.4% taurine content in the diet was required for optimum growth of juvenile flounder. A positive linear relationship was noted between the content of taurine accumulated in the muscle, liver and brain and the level of taurine in the diet. However, there was no increased taurine content in tissues of fish fed the cystine-supplemented diet. In contrast, the fish fed control and cystine-supplemented diets showed higher contents of cystathionine in the tissues. The concentration of cystathionine in tissues rapidly decreased with an increase of taurine in the diet. It was also observed that for each of the dietary groups, a trace amount of taurine was excreted. These results suggest that the taurine content in the diet affects the sulfur amino acid metabolism of juvenile Japanese flounder, and indicate that juvenile flounder are unable to biosynthesize taurine from cystine.     

Modeled transport of winter flounder larvae spawned in coastal waters of the Gulf of Maine

Winter flounder, Pseudopleuronectes americanus, from southern New England and the Gulf of Maine were historically considered to be obligate estuarine spawners. However, recent experiments and observations document that winter flounder in the Gulf of Maine also utilize coastal waters for spawning. An individual‐based modeling approach was used to investigate the transport of winter flounder larvae from three hypothesized coastal spawning grounds in the Gulf of Maine. Transport success rates were greatest for larvae released from Ipswich Bay, intermediate for Stellwagen Bank and least successful for those released from Jeffreys Ledge. There was substantial interannual variability in larval transport and geographic patterns of potential connectivity. Furthermore, the date of spawning had an important influence on transport success. Model results suggest that certain coastal spawning grounds used by winter flounder may serve as an important source of larvae to estuaries and nearshore nursery areas. The potential influx of coastal spawned larvae could have implications for the resilience, productivity and gene flow in local populations. Model results provide further support for the conclusion that winter flounder in the Gulf of Maine may not be solely dependent upon estuaries for spawning. Results also suggest that coastal spawning groups should be considered explicitly in the management of winter flounder, and protected under Essential Fish Habitat regulations.     

Genetic characterization, morphometrics and gonad development of induced interspecific hybrids between yellowtail flounder, Pleuronectes ferrugineus (Storer) and winter flounder, Pleuronectes americanus (Walbaum)

Viable interspecific hybrids between yellowtail flounder (Pleuronectes ferrugineus, Storer) and winter flounder (Pleuronectes americanus, Walbaum) were produced by artificial insemination of yellowtail flounder eggs with winter flounder sperm. However, mean fertilization rate, hatching success and early survival up to 3 weeks post hatch were significantly lower than those of parental pure cross controls (P < 0.01). Overall, cytogenetic traits (karyological analysis and estimation of cellular DNA contents using flow cytometry) of hybrid flounder were intermediate between the two parental species. Microsatellite assay was used to distinguish the parental genomes in the hybrids; in most cases, one allele was specific to each of the parents. Morphometrics assessed by body proportions indicated that hybrids generally displayed a morphology intermediate between the maternal and paternal species. Interspecific hybrids exhibited abnormal and retarded gonad development in both sexes based on histological analysis of gonads from adult fish. The sterility of the hybrids presents a significant advantage for their use in aquaculture, as potential escapees would not be capable of reproducing in the wild and contaminating natural stocks.     

Effects of different dietary levels of L-ascorbyl-2-polyphosphate on growth and tissue vitamin C concentrations in juvenile olive flounder, Paralichthys olivaceus (Temminck et Schlegel)

This experiment was conducted to study the effects of different dietary levels of vitamin C, L‐ascorbyl‐2‐polyphosphate (ASPP), on growth and tissue vitamin C concentrations in juvenile olive flounder, Paralichthys olivaceus (Temminck et Schlegel). Fish were fed one of six semi‐purified diets containing an equivalent of 0, 25, 50, 75, 150, or 1500 mg ascorbic acid (AA) kg^?1 diet (C₀, C₂₅, C₅₀, C₇₅, C₁₅₀ or C₁₅₀₀) in the form of ASPP for 12 weeks. Weight gain (WG) and protein efficiency ratio (PER) of fish fed the C₀ diet were significantly lower than those of fish fed the other diets (P < 0.05), and WG and PER of fish fed the C₂₅, C₅₀ and C₇₅ diets were significantly lower than those of fish fed the C₁₅₀₀ diet (P < 0.05). Fish fed the C₀ diet exhibited vitamin C deficiency symptoms such as anorexia, scoliosis, cataract, exophthalmia and fin hemorrhage at the end of the 12‐week test. After 12 weeks of the feeding trial, AA concentrations from gill, kidney, and liver of fish fed the C₀, C₂₅, C₅₀ and C₇₅ diets were significantly lower than those of fish fed the C₁₅₀ and C₁₅₀₀ diets (P < 0.05). Based on broken line analyses for WG and PER, the optimum dietary levels of vitamin C were 91 and 93 mg AA kg^?1 diet respectively. These findings suggest that the dietary vitamin C requirement could be 93 mg AA kg^?1 diet to support reasonable growth, and greater than 150 mg AA kg^?1 diet may be required for AA saturation of major tissues for juvenile olive flounder under experimental conditions.