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.     

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.     

Growth of early juvenile winter flounder (Pseudopleuronectes americanus Walbaum)

Metamorphosis is a critical developmental stage in marine fish species, but mortalities are also elevated at the settling phase. Our objective was to evaluate the effects of different rearing systems and larval diets on survival, general condition and pigmentation of newly settled winter flounder (Pseudopleuronectes americanus) juveniles. At settlement, winter flounder juveniles were reared in two different types of tanks (plankton kreisel and rectangular raceway). The early juveniles were followed for 1 month and their condition was evaluated using biochemical indices. Juveniles did not show signs of fin rot and pigmentation was well developed, but they had different RNA/DNA ratios. This study highlights the fact that rearing conditions at the onset of the juvenile stage may be critical for successful juvenile production.     

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.     

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.     

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.     

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.     

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.     

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     

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.     

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.     

匙吻鲟仔鱼期饵料的研究

鱼类早期生长的细微差别对其存活以及整个生活史阶段的生长,有着不可估量的影响,而摄食是影响仔鱼期生长的最重要的外界影响因子之一。在水温19．5～20．5℃下,对匙吻鲟（Polyodon spathula）仔鱼的开口时间、适宜开口饵料和投饵率进行了研究。结果表明,匙吻鲟仔鱼在6日龄初次摄食,摄食率在7—8日龄迅速升高,9—10日龄达到100％,11-13日龄略有降低,14-15日龄迅速降低,不可逆点（PNR期）出现在15日龄;以轮虫作为开口饵料,仔鱼的存活率和生长优于丰年虫和颗粒饲料组;养殖水体中轮虫密度在120—150个／mL可满足仔鱼的生长需求。     

Marine raw material choice,quality and weaning performance of Ballan wrasse (Labrus bergylta) larvae

Previous results show that weaning success of Ballan wrasse larvae greatly depends on the quality of the dietary marine raw materials. In the present study, six moist or agglomerated experimental weaning diets containing different combinations of high‐quality marine raw materials, being fish meal (FM), cod muscle meal (CMM), shrimp meal (SM) and krill hydrolysate (KH), were tested in a 2‐month weaning trial with Ballan wrasse larvae of 34.5 mg initial body weight. Larvae performance was good in all dietary treatments except those fed diet D1 containing only FM. The Ballan wrasse larvae fed weaning diets D4 and D5 containing FM and either SM or KH, respectively, had the highest final body weight (0.7 g) but also the highest mortality (50%). Best weaning survival (77%) was obtained using the dry agglomerated diet D3 containing CMM and SM. During the first month, fish survival correlated positively with dietary free amino acid and soluble protein levels and negatively with the combined levels of dietary lipid oxidation metabolites and ethoxyquin. During the second month, mortality rates were lower in all treatments. Fish larvae final body weight correlated negatively with total dietary fatty acids and positively with dietary cholesterol, phosphorous and DHA/EPA ratio.     

Gelatin-walled microencapsulated diet for larval shrimp (Penaeus japonicus Bate) manufactured using the fluidized bed coating process

The gelatin-walled microencapsulated feed for larval shrimp (Penaeus japonicus) was produced using the fluidized bed coating process. The microencapsulated diet showed no significant agglomeration in the coating process and the diameters of the microdiet were in a normal distribution. Scanning electron microscopy microphotographs showed the appearance of a microencapsulated diet with a uniform surface and a continuous film around the core. The retention efficiency of vitamin C was 88.2% in the coating process. The inclusion efficiency, lipid encapsulation efficiency and nitrogen retention efficiency of the microdiet were 92.2 ± 1.6%, 76.8 ± 4.1% and 60.6 ± 5.2% respectively. The mysis α of P. japonicus were reared for 20 days to the later larval stage. The wet weight of larval shrimp increased 300.0% in the microdiet from 10 up to 30 day after hatching. The wet weight and the total length of larvae were greater in co-feeding compared with the control (P<0.05), but there was no significant difference in larval survival between co-feeding and control (P>0.05). There were significant differences (P<0.05) in the wet weight, total length and survival in the microdiet compared with the control. The results indicated a huge potential for the use of the gelatin-walled microencapsulated diet for the partial and total replacement of live food for larval shrimp.     

Early introduction of an inert diet and unenriched Artemia enhances growth and quality of Atlantic cod (Gadus morhua) larvae

The effects of two weaning diets and different weaning protocols on growth, survival, skeletal deformity and gut morphology of Atlantic cod larvae were studied in four groups from 16 to 45 days posthatch (dph). Cod larvae in groups 1 (early weaning with control diet) and 2 (early weaning with experimental diet) were used to evaluate the effects of different polar lipid content of weaning diets on larval and juvenile performance. Cod larvae in groups 2, 3 (early weaning with experimental diet + cofeeding with Artemia) and 4 (earlier weaning with experimental diet and earlier cofeeding with Artemia) were used to evaluate the effects of early introduction of dry diet and Artemia. From 45 to 170 dph, cod juveniles from all four groups were reared using a standard feeding protocol. No significant differences in growth, survival, deformities and gut morphology were found between cod larvae and juveniles from groups 1 and 2. Cod larvae fed on cofeeding regime with Artemia nauplii (groups 3 and 4) were bigger and had lower frequencies of jaw and neck deformities and higher foregut microvillus circumference than cod larvae from group 2. Our results demonstrate the importance of proper weaning protocols in producing better quality cod juveniles.     

Evaluation of weaning performance of California halibut (Paralichthys californicus) larvae using growth,survival and digestive proteolytic activity

This study evaluated weaning success of California halibut, Paralichthys californicus, larvae onto a microdiet at various stages of development utilizing growth, survival and digestive enzyme activity. Weaning onto a microdiet was evaluated at 16, 26, 36 and 46 days posthatch (dph). Alkaline and acid proteases and leucine aminopeptidase activities were measured after weaning and compared between the weaned treatment and Artemia‐fed controls. Survival was significantly lower in the microdiet‐fed treatments compared to the control groups. Growth was significantly reduced in all weaning treatments compared to the control, except for the 46 dph group. No differences in enzyme activities were detected between treatment and diet at 16 and 26 dph; however, activities were higher for the microdiet‐fed larvae at 36 and 46 dph. This study demonstrates that California halibut larvae possess a differentiated and effective digestive system early in development and can be weaned with relative success (>40% survival) before completion of the metamorphosis (i.e., 36 dph). The lack of weaning success at an early date cannot be entirely because of the absence of a functional stomach but could be related to, among other factors, the low‐microdiet ingestion rates observed and higher leaching of smaller microdiets.     

Modelling the transport of lobster (Homarus americanus) larvae and postlarvae in the Gulf of Maine

We used a coupled physical–biological model to examine potential distances between hatching and settlement locations for lobsters in the Gulf of Maine. The physical model is based on a finite-element mesh and climatological averages of the seasonally evolving temperature and density fields. Larval trajectories from coastal and offshore hatching sites (21–224 m deep) were calculated for early, middle and late-season hatching by coupling temperature-dependent development rates and depth (the biological model) to the circulation. Model results showed large spatial differences in larval development times (from 18 to 38 days) and distances transported (19–280 km) for the early hatch. Development time and transport decreased markedly by mid-season at most sites, but strong spatial differences persisted. The eastern Maine coast appears to experience stronger removal and less resupply of larvae than other regions, consistent with observed lower recruitment. Inverse solutions of the model for larvae arriving in mid-coastal Maine indicate that they originate from a broad section of the eastern coast 'upstream', with those nearest the shoreline generally travelling the shortest distances. The postlarval stage is neustonic (living near the surface), and a simple inverse model demonstrates that a diurnal coastal sea breeze can contribute substantially to inshore movement during this final planktonic stage. Thus, offshore reproduction may be linked to inshore recruitment.     

Determination of appropriate age and stocking density of vundu larvae, Heterobranchus longifilis (Valenciennes 1840), at the weaning time

Two experiments were conducted in order to determine the appropriate age and stocking density of vundu catfish Heterobranchus longifilis at the weaning time. In the first experiment, five triplicate groups of 100 larvae (initial mean weight=3.4 mg) per aquaria were stocked from first feeding [day 3 post‐hatch (p.h.)] to day 30 p.h., and then weaned, on days 3 (W3), 5 (W5), 8 (W8) and 14 (W14), and an unweaned group (An). Significant differences were observed in growth, survival, cannibalism, coefficient of weight variation and body composition among larvae weaned at different ages and the control group. The later the larvae were weaned, the better were the growth performances [final mean weight: from 65.1 to 201.1 mg and specific growth rate (SGR): from 11.0 to 15.2% day^?1] and the survival (from 36.5% to 74.3%). The experiment with stocking densities of 5, 10, 25 and 50 larvae L^?1 showed that increasing the stocking density decreased growth performances and weight variation but improved the survival rate of larvae. The best growth performances (SGR=13.4 and 11.4% day^?1) with the lowest survival rates (70.3% and 77.3%) were observed in larvae stocked at densities of 5 and 10 larvae L^?1 respectively.     

Replacement of live food with a microbound diet in feeding Litopenaeus setiferus (Burkenroad) larvae

Microbound feeds have been well accepted by shrimps and farmers in many penaeid shrimp hatcheries. The present study focused on an adequate level of replacement of Artemia nauplii and microalgae by a microbound diet for rearing Litopenaeus setiferus (Burkenroad) larvae. A microbound diet (MBD) consisting of fishmeal, squid meal, shrimp meal, yeast meal and soybean meal was used. The first experiment was designed to obtain the optimum level of MBD to complete the live feeding schedule, from Protozoea (PZ_III) to Mysis (M_III). The experimental levels of the microbound diet tested were 2, 4, 6 and 8 mg MBD L^?1 day^?1. The next step was to determine the Artemia nauplii replacement level from PZ_I to M_III by MBD. These experiments were carried out either in the presence (Experiment 2) or in the absence of algae (Experiment 3). Four replacement levels were tested: 0% (4 mg MBD L^?1 day^?1: 1 Artemia nauplii mL^?1), 40% (5.5 mg MBD L^?1 day^?1: 0.6 Artemia nauplii), 60% (6.5 mg MBD L^?1 day^?1: 0.4 Artemia nauplii) and 100% (8 mg MBD L^?1 day^?1: 0 Artemia nauplii). In all experiments growth, survival, development, quality index (QI) and performance index (PI), were used to determine the optimum concentration of microbound diet. Results showed that 6 mg MBD L^?1 day^?1 can be recommended as a complement to live food for L. setiferus larvae from PZ_III to M_III. In the presence of algae, maximum growth and survival may be obtained in 40–60% (5.5–6.5 mg MBD L^?1 day^?1) of Artemia nauplii replacement levels. In the absence of algae, the Artemia nauplii replacement resulted in slower development, less salinity resistance, lower growth and lower survival than was obtained in larvae fed with algae.