Experimental infection of Atlantic halibut, Hippoglossus hippoglossus L., yolk-sac larvae with infectious pancreatic necrosis virus: detection of virus by immunohistochemistry and in situ hybridization

Three different concentrations (10⁷, 10⁵ and 10³ TCID₅₀ ml^-1) of infectious pancreatic necrosis virus (IPNV) serotype Sp isolated from Atlantic halibut, Hippoglossus hippoglossus L., were used to bath-challenge Atlantic halibut yolk-sac larvae. The larvae challenged with 10⁷ TCID₅₀ ml^-1 suffered significantly higher cumulative mortality than the other challenged groups and the control group, and affected individuals displayed necrosis of the intestine, liver and kidney. In larvae from the groups challenged with 10⁷ and 10⁵ TCID₅₀ ml^-1, IPNV was detected by immunohistochemistry and in situ RNA/DNA hybridization in the intestine, liver and kidney. In addition, some individuals stained IPNV-positive in the heart and eye/brain region. Detection by in situ hybridization did not appear to be more sensitive than immunohistochemistry. However, background staining was virtually absent in comparison with immunohistochemistry, and the staining seemed to be more distinctly localized to the cytoplasm of infected cells. The results show that farmed halibut yolk-sac larvae can be infected by IPNV immediately after hatching, with resulting high mortality. As the larvae are not immunologically mature at this stage of development, vaccination is not recommended.     

The effect of light on activity and growth of Atlantic halibut, Hippoglossus hippoglossus L., yolk-sac larvae

The growth and activity of Atlantic halibut, Hippoglossus hippoglossus L., yolk-sac larvae exposed to light of differing intensities and wavelengths were investigated every fifth day. The experiments were conducted at 6 ^oC from day 1 until day 34 post hatch. Four intensities of constant white light (2.0, 0.3, 0.03 and 0.005 μ Em^?2 s^?1, λ_max 590 nm), and constant coloured light of equal intensities (0.03μ Em^?2s^?1) in the blue, green and red spectrums (λ_max 450, 560 and 670 nm, respectively) were used. In addition to a control treatment in constant darkness, one treatment was incubated in a 12:12 h light: dark photoperiod. The light treatments did not have any discernible effect on the total length, myotome height, dry weights or yolk conversion efficiencies. The most intense white light resulted in an increased activity on days 24 and 30 post hatch, resulting in a temporarily reduced length and myotome height for the larvae in these groups compared to the other treatments. Larvae from all treatments were of the same size 34 days post hatch. The dry weights of the larvae and yolk-sacs were unaffected by light treatment. The activity increased independently of light treatment until 120 degree-days, and thereafter, the strongest white light resulted in an temporarily increased activity. The distribution of activity changed independently of light regime in the beakers during development.     

Time of first feeding of Atlantic halibut, Hippoglossus hippoglossus L., larvae

Atlantic halibut, Hippoglossus hippoglossus (L.), eggs originating from one female were evenly distributed between four silos (4.8 m³) shortly prior to hatching. At days 30, 35, 40 and 44 after hatching [i.e. 200, 230, 260 and 290 day-degrees (days^o)], the larvae were successively collected and transferred to indoor start feeding tanks, and larvae were offered a diet of instar II Artemia nauplii which had been enriched short time (24 h). A significant correlation was found between the age of the larvae and onset of first feeding. The larvae transferred to start-feeding incubators at 290 days^o were able to capture Artemia only a few hours after transfer, whereas it took 6 days for the larvae transferred at 200 days^o to reach a corresponding ingestion level. Larval growth was also positively correlated to both larval age and prey consumption. However, there were no differences in survival between the larval groups.     

Atlantic halibut, Hippoglossus hippoglossus L., larvae cultivation literature, including a bibliography

The first attempts to rear Atlantic halibut, Hippoglossus hippoglossus L., larvae were carried out in Norway in the period from 1974 to 1980, when ripe adult specimens of Atlantic halibut were net-caught, and stripped for eggs and milt. Both incubation of yolk-sac larvae and first-feeding were carried out in large submerged plastic bags and the larval food consisted of natural zooplankton collected from surrounding lagoon water. This semi-extensive production method was further developed and led to the establishment of several commercial production trials by the end of the 1980s. During recent years, research has been focused on intensive methods for first-feeding and the combined effort of several research institutes has resulted in a reliable production method. During the late 1980s and early 1990s, the main research activity was focused on the biology of and rearing techniques for eggs and yolk-sac larvae. These techniques provided satisfactory yields for several years. However, during the past few years, a certain decrease in survival through the yolk-sac stage has been experienced at several hatcheries. Since the early 1990s, the first-feeding period has represented the bottleneck in the development of a reliable rearing method. The main effort has been concentrated on system configuration and on improving live prey quality. In the future, new feeding strategies including further improvement of live prey (i.e. Artemia), the use of copepods and early weaning onto a formulated diets should be emphasized. Further research on hygiene and technological improvements is needed to increase growth and survival through metamorphosis.     

A tank design for first feeding of Atlantic halibut, Hippoglossus hippoglossus L., larvae

An intensive method of juvenile Atlantic halibut, Hippoglossus hippoglossus (L.), production has been under development over the last decade because of the problems associated with the extensive method. The lack of initiation of feeding behaviour has been the main obstacle for successful indoor rearing under artificial light and feeding conditions. In the present paper, an intensive method for the first feeding of halibut larvae is described and verified by practical feeding trials. The method involves circular 1.5-m³ indoor tanks with a peripheral ring-shaped cover, the use of continuous light, UV-A radiation during the first 24 h, central up-welling of water made by aeration and the use of microalgae in the rearing water. The single most important factor in such systems is to maintain a current pattern which allows the larvae to orientate and position themselves to face the water current for easy capture of prey.     

Experimental infection of eggs and yolk sac larvae of halibut, Hippoglossus hippoglossus L.

Abstract Scanning electron micrographs of halibut eggs with an epiflora dominated by Flexibacter sp., showed ulcerations colonized by large numbers of bacteria. The chorion was dissolved in most ulcerations and the zona radiata was severely damaged. Infection experiments showed that exposure to these bacteria caused high mortalities at the late egg stage, hatching and the early yolk sac stage. Eggs exposed to three different Vibrio spp. showed different mortality patterns, with low mortality at hatching, followed by a continuous high mortality throughout the yolk sac stage. Mortality in the uninfected control group was low throughout the experiment. Transmission electron microscopy of the Vibrio -infected larvae showed bacteria present in the gill, heart and frontal yolk sac regions.     

A formulated diet for Atlantic halibut (Hippoglossus hippoglossus, L.) larvae

A diet for Atlantic halibut-larvae was formulated taking into account the fact that marine-fish larvae have a limited ability to assimilate protein and lipid. Dietary protein consisted of a free amino-acid premix (7.2% of crude protein), predigested-squid mantle (7.2%), squid mantle (8.6%) and cod-muscle mince (77.0%). Lipid sources were soyabean lecithin (33% of crude lipids), crude phospholipids extracted from cod roe (10%) and sardine oil (57%). Larvae were weaned onto the experimental diet at wet-body weights of 0.07, 0.10 or 0.16 g, respectively. The experimental diet was fed for 31, 25 or 17 days, respectively, and the experiment was terminated on the same calendar day for all groups. A control group was fed with Artemia nauplii enriched with DHA Selco™ from 0.07 g. Survivals ranged from 78% in larvae transferred at 0.10 g to 96% in those transferred at 0.16 g and in the control group. Daily specific-growth rates (SGR) were 3.1 ± 0.07, 3.3 ± 0.11 and 2.2 ± 0.01% day⁻¹ in larvae transferred at 0.07, 0.10 and 0.16 g, respectively, while growth in the control group was 5.1% day⁻¹. It was concluded that weaning of Atlantic-halibut larvae is feasible from 0.7 g (approximately 20 days post first-feeding) when the formulated diet contains predigested protein and ample amounts of phospholipids.     

Folate in eggs and developing larvae of Atlantic halibut, Hippoglossus hippoglossus L.

Folate mobilization from the yolk compartment during larval development was studied by analysing the folate concentration in whole body, embryo and yolk in a single batch of Atlantic halibut, Hippoglossus hippoglossus L., eggs and larvae that showed successful fertilization and development. There was a net loss of approx. 50% of folate from yolk during endogenous feeding. Further, only 23% of the decrease in yolk folate was retained in the larval body. The data suggest a need for folate for metabolic and growth purposes during embryogenesis of approximately 2 μg g^?1 weight gain. Relative to these data and published folate requirement for cold‐water species, batches of egg from 16 Atlantic halibut brood fish contained variable and, for some batches, critically low levels of folate. This may constitute a potential problem for larval development until start feeding.     

Design and operation of Atlantic halibut, Hippoglossus hippoglossus L., egg incubators

An egg incubator system specially designed to meet the requirements of Atlantic halibut, Hippoglossus hippoglossus L., eggs is described. Compared to other pelagic marine fish eggs, halibut eggs are frequently found to be heavy and lacking in buoyancy in seawater incubators. The biological features of halibut eggs which explain the choice of system design are discussed. In addition to the biological requirements, the system described is designed to meet the demands of commercial hatcheries where handling of large egg masses with a minimum of labour is needed. The incubation method has proved by experience to give high survival rates throughout egg development.     

A correlation between phototactic response and first-feeding of Atlantic halibut (Hippoglossus hippoglossus L.) larvae

The correlation between positive phototaxis and feeding incidence at first‐feeding for groups of Atlantic halibut larvae ranging in age from 210 to 240 degreedays, post hatch, was examined. Phototactic response was measured as the fraction of larvae that responded by horizontal swimming towards a light source, and the median travel distance for the responding fraction of larvae. Within the ranges investigated, larval age, size or proportion of deformed larvae had no significant effect on the phototactic response, or on the feeding incidence (deformed larvae excluded from analysis). The fraction of larvae responding phototactically and the median travelling distance for responding larvae were significantly correlated. Feeding incidences after 24 and 48 h were also significantly correlated with both measures of phototactic response. The possibility of using phototactic response in quality assessment is discussed.     

Feeding strategies to achieve correct metamorphosis of Atlantic halibut, Hippoglossus hippoglossus L., using enriched Artemia

Atlantic halibut, Hippoglossus hippoglossus L., larvae were reared under four different Artemia feeding regimes for 40 days from day 20 post first feeding. Three Artemia enrichments were used: Super Selco? (SS), AlgaMac 2000? (AM) and a particulate mix of AlgaMac 2000? with tuna orbital oil (TOO/AM). The SS and AM Artemia were tested in three different combinations: (1) a 1:1 ratio continuously to day 60 (AM/SS); (2) a 1:1 ratio to day 41, then AM only (AM/SS-41): and (3) AM only to day 41, then a 1:1 ratio (AM-41). The fourth treatment comprised TOO/AM Artemia, which was fed continuously. At day 60 post first feeding, measurements were made of survival and growth rates, postmetamorphic characteristics (i.e. eye migration and pigment distribution), and lipid composition. The mean survival rates ranged from 65.1% to 84.5%. Specific growth rates varied from 7.64 to 8.13. The eye migration indices were between 2.3 and 2.6. These parameters did not differ significantly among treatments (P 0.05). A significantly greater proportion of fry (P<0.05) exhibited‘perfect metamorphosis’(correct pigment distribution and complete eye migration) in the AM/ SS-41 and AM-41 treatments (59.8 ± 3.03% and 54.6 ± 1.08%, respectively) compared to the AM/SS and TOO/AM treatments (35.9 ± 4.02% and 39.9 ± 6.43%, respectively). The fatty acid compositions of livers and eyes varied according to feeding regime, but did not correspond to the metamorphosis characteristics of the fry.     

Tissue tropism of nervous necrosis virus (NNV) in Atlantic cod, Gadus morhua L., after intraperitoneal challenge with a virus isolate from diseased Atlantic halibut, Hippoglossus hippoglossus (L.)

Atlantic cod, Gadus morhua , averaging 100 g, were experimentally challenged by intraperitoneal injection of nervous necrosis virus (NNV) originating from Atlantic halibut. Cod tissues, including blood, gill, pectoral fin, barbel, ventricle, atrium, spleen, liver, lateral line (including muscle tissue), eye (retina) and brain, were sampled at day 25 and 130 and investigated by real-time RT-PCR for the presence of NNV. Relative quantifications at day 130 were calculated using the 2^−ΔΔCt method. Immunosuppression by injection of prednisolone-acetate was introduced for a 30-day period, and tissue sampled at day 180 and relative quantification estimated. No mortality or clinical signs of disease were observed in the challenged group. The challenge resulted in detection of NNV in blood, spleen, kidney, liver, heart atrium and heart ventricle at day 25, and by the end of the experiment NNV showed a clear increase in brain and retina, suggesting these to be the primary tissues for viral replication. There was no increase in the relative amount of NNV in blood, atrium, ventricle, spleen, liver and kidney. Corticosteroid implants resulted in a weak increase in virus RNA in spleen, kidney, liver and brain. These findings suggest that Atlantic cod is susceptible to infection with NNV from halibut. The observed tissue tropism patterns suggest an initial viraemic phase, followed by neurotrophy. Head-kidney is the best tissue identified for possible NNV detection by non-lethal biopsy, but detection was not possible in all injected fish.     

Pigmentation and eye migration in Atlantic halibut (Hippoglossus hippoglossus L.) larvae: new findings and hypotheses

Atlantic halibut juveniles, which have been fed Artemia during larval development, frequently demonstrate malpigmentation and impaired eye migration. This is in contrast to the high percentage of normally developed larvae fed copepods, reared under similar conditions. Nutrition is therefore an important component influencing larval development. Analyses of the nutrient composition of Artemia and copepods show that Atlantic halibut larvae fed Artemia probably receive sufficient amounts of vitamin A by converting canthaxanthin, while iodine may be deficient, possibly leading to interrupted thyroid hormone synthesis. An unbalanced fatty acid composition, such as high levels of arachidonic acid and low levels of docosahexaenoic acid, can be another limiting factor in Artemia. Vitamin A, fatty acids and thyroid hormones have all been shown to affect pigmentation in flatfish. They are ligands to nuclear receptors, thyroid hormone receptors, retinoic acid receptors, retinoic X receptors and peroxisomal proliferator‐activated receptors, which are members of the superfamily of steroid hormone receptors. The receptors interact with each other to promote gene expression that modulates proliferation and differentiation of cells. Our hypothesis is that these interactions are important for development during flatfish metamorphosis. Very little data exist on the topic of impaired eye migration. However, energy limitation, iodine deficiency and an unbalanced fatty acid composition have been proposed as possible explanations. Here, we review the literature on development of pigment cells and the possible mechanisms behind the effects of vitamin A, fatty acids and thyroid hormone on pigmentation and eye migration during development of Atlantic halibut larvae.     

Effect of predigested protein on growth and survival of Atlantic halibut larvae (Hippoglossus hippoglossus L.)

In this study Atlantic halibut (Hippoglossus hippoglossus L.) larvae (0.12 ± 0.04 g) were, from day 40 post first feeding, offered six diets in which 10% or 30% of the dietary protein was hydrolysed with (a) pepsin (P), (b) pepsin + trypsin (PT) or (c) pepsin + trypsin + chymotrypsin (PTC). In addition, a diet without hydrolysed protein was offered, and enriched Artemia was fed as control. The amount of soluble protein increased progressively with the enzyme treatments P, PT and PTC and with higher inclusion levels of hydrolysed protein. Survival was highest among the larvae offered Artemia (83 ± 0%) or the diet 10P (10% pepsin hydrolysed protein; 67 ± 4%). The diet 10P supported survival significantly better than the more hydrolysed diets 10PTC, 30P, 30PT and 30PTC, but not significantly better than the non‐hydrolysed diet and 10PT. Specific growth rate (SGR) was 1.76 ± 0.20 in average for all groups of larvae and was not significantly affected by the diets. Still, the larvae offered pepsin hydrolysed diets tended to have better growth (2.10 ± 0.05 SGR; P < 0.06) than the larvae offered the other hydrolysed diets. The larvae offered the formulated diets did not differ in chemical composition.     

Compartmental changes in the contents of total lipid, lipid classes and their associated fatty acids in developing yolk-sac larvae of Atlantic halibut, Hippoglossus hippoglossus (L.)

Total lipid, lipid classes and their associated fatty acids have been measured in whole halibut, Hippoglossus hippoglossus (L.) larvae and in dissected animals separated into yolk and body compartments. At hatching the larval body contained 17 μg ind^?1 of lipid (11% of larval body dry weight), while the yolk contained 190 μg ind^?1. Phosphatidylcholine (PC) accounted for 57% of total yolk lipids while phosphatidylethanolamine (PE), triacylglycerol (TAG), cholesterol and sterol ester (SE) accounted for 12%, 12%, 9% and 6% respectively. The main fatty acids in the PC fraction were 22:6n-3 (25.6 μg ind^?1), 16:0 (19.2 μg ind^?1) and 20:5n-3 (12.6 μg ind^?1). Between hatch and 200 day-degrees post hatch (D°PH) a net decline in total lipids of 29% was seen. There seemed to be some, but relatively minor, changes in the relative composition of lipids in the yolk throughout development, which are indicative of a non-selective endocytotic bulk uptake of lipids from the yolk. Towards first-feeding there was a selective catabolism of PC and a net synthesis of PE in the developing body, resulting in a shift in the lipid class composition in the body compared with that of the yolk. The fatty acids released from lipid hydrolysis were mainly used as energy substrates by the growing halibut larvae; 22:6n-3 was quantitatively one of the most important fatty acid fuel in energy metabolism. At the same time 38% and 23% of the 22:6n-3 released from PC was retained by the PE and neutral lipids in the growing larval body respectively. Except for 20:5n-3 (2%, 14%) no similar retention was seen in any of the other fatty acids. The observed net synthesis of PE in developing yolk-sac larvae of Atlantic halibut and the preferential retention of 22:6n-3 into it, increasing from 28% at hatching to 45% at 200 D°PH, may point to a high biological value of this compound.     

Effects of light administration and algae on first feeding of Atlantic halibut larvae, Hippoglossus hippoglossus (L.)

The aim of the study was to investigate the effects of two light regimes that independently had shown positive effects on feeding and growth in cultures of Atlantic halibut, Hippoglossus hippoglossus (L.), larvae. The regimes were low-intensity overhead light and submerged light at intermediate light intensities. Secondly, an alleged beneficial effect of algae was investigated. An experiment was designed to include four different regimes in the larval cultures: low-intensity overhead light with and without algae (Tetraselmis sp.), and submerged light with and without algae. The results showed that submerged light was superior to overhead light with respect to larval growth, survival and feeding incidence. It was further indicated that algae improved larval growth and survival, but no effect was shown on feeding incidence. There was, however, no interaction between the effects of algae and those of the light regime. The causal effect of the algae may be ascribed to indirect factors, such as light attenuation as well as a direct and indirect nutritional effect.     

Suitable methods for cryopreservation of semen from Atlantic halibut, Hippoglossus hippoglossus L.

Decrease in the quality and quantity of Atlantic halibut, Hippoglossus hippoglossus L., semen towards the end of the reproductive season hampers production of good-quality embryos. Therefore, cryopreservation of spermatozoa is a method showing potential to facilitate controlled reproduction in Atlantic halibut. The present study aimed at establishing the appropriate cryopreservation procedure. We tested 20 extenders composed of four various diluents and five cryoprotectants (DMSO, DMA, methanol, propylene glycol, and glycerol) to determine the best extender. Then, we examined cryopreservation quality using various methods of loading and various volumes of cryopreserved samples. In most of the tested variants, sperm diluted with an extender showed high motility after 24-h incubation despite the high osmotic pressure of the extender. Modified turbot extender (MTE) was the best of the tested diluents, securing the highest post-thaw motility (P < 0.05), and DMSO, DMA, and methanol were the best cryoprotectants (P < 0.05). There was no significant effect of 15-min equilibration of semen in MTE-based extenders prior to freezing on post-thaw motility (P > 0.05). MTE-based extender was chosen as the most suitable. Semen cryopreserved in straws, Eppendorfs or Ziploc bags in volumes ranging from 0.25 to 20 ml showed similar high fertilization ability. Survival of larvae produced with the cryopreserved sperm did not differ from controls produced with freshly collected sperm. Motility 3 h after thawing was high but depended on the type of cryoprotectant and the volume of cryopreserved sperm (P < 0.05). The developed cryopreservation procedure has been applied at our Atlantic halibut breeding station for seed production.