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In marine fishes of commercial interest, defining habitat use and migration strategies through ontogeny can help better understand the structure and dynamics of harvested populations and guide their management. The present study relied on otolith chemistry to identify three contingents within the Atlantic halibut (Hippoglossus hippoglossus) stock in the Gulf of St. Lawrence (GSL). We differentiated two chemical signatures from otolith edges, one for shallow (<100 m) and another one for deep (>100 m) waters. By identifying transitions between the deep and shallow habitats, we found that most halibut display migrations from the deep waters to shallow waters during the first 3 years of life. After reaching maturity, most halibut distributing in northern regions of the GSL became full-time residents in deep areas of the GSL. In contrast, halibut found in summer on the shallow plateau of the southern GSL displayed migrating behaviour between shallow (summer) and deep (winter) waters throughout their lives, either on an annual or irregular basis. Overall, our results demonstrate that otolith chemical signatures serve as natural markers of geographically distinct marine environments, facilitating the identification and reconstruction of environmental histories of long-lived marine fishes. [ABSTRACT FROM AUTHOR]

The data-limited nature of Atlantic halibut (Hippoglossus hippoglossus) in U.S. waters hampers evaluation of what may be a slow but steady rebuilding pattern. Here, we collaborate with the commercial fishery to design and implement a multi-gear sampling program that collected 100s of biological samples from throughout the Gulf of Maine in a five-year period, 2014-2018. Examination of sectioned otoliths revealed a maximum age of 12 years (females) and 13 years (males); in comparison, Atlantic halibut as old as 40-50 years have been collected elsewhere in the western North Atlantic. Growth modeling confirmed sexual dimorphism, with a larger asymptotic length (L8) for females (214 cm fork length [FL]) than males (195 cm FL). Estimates of median female length at maturity, L50, of 128 cm FL (124-132 cm, 95% confidence limits), and median female age at maturity, A50, of 9.6 years old (9.0-10.8 years), were longer and older than previous reports for the Gulf of Maine, likely resulting from our use of histological instead of macroscopic methods to classify maturity. Histology demonstrated that vitellogenesis initiated in individuals in spring, nearly a year prior to spawning, which allowed us to identify first-time (primiparous) spawners and provided the first potential evidence of skip spawning for this species. Finally, an index was developed to track the proportion of potentially mature females in the fishery, which showed an increasing trend; this qualitative tool may prove useful in a data-limited environment for evaluating the relative stock status of Atlantic halibut. [ABSTRACT FROM AUTHOR]

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Atlantic halibut (Hippoglossus hippoglossus, Linnaeus, 1758) are a data-poor stock within the waters of the United States. This study evaluated the use of otolith measurements to back-calculate lengths of Atlantic halibut at previous ages. Back-calculations have proven useful for estimating length at age and growth rates of other species. To the best knowledge of the authors, this study is the first to document the use of this method for Atlantic halibut. Otolith back-calculations rely on a few key assumptions, such as proportionality of fish length and otolith length, which are not always met. This study shows that back-calculations using the Fraser-Lee method can provide reasonable estimates of Atlantic halibut length at previous ages, especially when samples from young halibut are included to improve estimates of the intercept of the linear regressions. Based on back-calculated estimates, female and male halibut in the Gulf of Maine showed different growth rates after age five. There was no evidence of changes in growth rates over an approximately 15 year time period. Halibut caught in the Gulf of Maine and on the neighboring Scotian Shelf showed some differences in growth rates; however, the results did not support strong conclusions about differences between the two regions as the direction of the differences was not consistent between the sexes and previous tagging studies have shown extensive movement between the two areas. The finding of reasonably accurate back-calculated lengths at previous ages is important for this data-poor species, as back-calculations increase the amount of information that can be obtained from otoliths.

Characterizing migratory behaviours contributes to the sustainable management of marine fishes by resolving stock structure and identifying the timing and locations of events within fish life cycles. The migratory behaviour of Atlantic halibut (Hippoglossus hippoglossus) in the Gulf of St. Lawrence (GSL), Canada was characterized over an annual cycle using pop-up satellite archival tags (n=15). Daily probability density functions of individual halibut positions were estimated using a geolocation model specifically developed to track demersal fish species in the GSL. Reconstructed migration routes (n=8) revealed that Atlantic halibut displayed seasonal migrations, moving from deeper offshore waters in the winter to shallower nearshore waters in the summer. Variability in migratory behaviours was observed among individuals tagged at the same location and time. One individual resided year round in the vicinity of the tagging site, three individuals displayed homing behaviour, and four individuals did not return to the tagging site. The identification of presumed spawning rises for two individuals suggested that spawning of Atlantic halibut occurred in the GSL. Although based on a limited number of individuals, these results suggest that Atlantic halibut in the GSL forms a philopatric population, supporting the current separate management of this stock from the adjacent Scotian Shelf and southern Grand Banks stock. [ABSTRACT FROM AUTHOR]

Juvenile Atlantic halibut (~100 mg, Hippoglossus hippoglossus) were exposed to Vibrio proteolyticus, a Vibrio spp. isolate, Photobacterium damselae ssp. damselae and five different isolates of Aeromonas salmonicida ssp. achromogenes via an hour-long bath immersion to ascertain their variation in pathogenicity to this fish species. Results were analysed using Kaplan-Meier survival analysis. Analysis of the data from challenges using A. salmonicida ssp. achromogenes revealed three survival values of zero and a spread of values from 0 to 28.43. Challenges using a Vibrio spp isolate, V. proteolyticus and P. damselae resulted in Kaplan-Meier survival estimates of 31.21, 50.41 and 57.21, respectively. As all bacterial species tested could induce juvenile halibut mortalities, they must all be considered as potential pathogens. However, the degree of pathogenicity of A. salmonicida is isolate dependent. [ABSTRACT FROM AUTHOR]

One of the main problems facing Atlantic halibut hatcheries is the high mortality in the early stages of larval development. Several factors could be involved, for example: water quality, diseases or abnormalities, such as deformities occurring in the yolk sac larvae prior to exogenous feeding. The aim of this study was to identify differences in bacterial flora associated with yolk sac larvae with oral deformity. We also aimed to establish whether there is any relationship between bacterial strains and the "gaping jaws" syndrome. During our study, 74 bacterial isolates were obtained using three different nutrient media: Marine Agar, R2A and TCBS. Some of these bacteria were characterized using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and 16S rRNA sequencing. The immune response in larvae exhibiting the "gaping jaws" condition was measured by real time PCR. Our results showed significant differences in bacterial flora between normal and gaping larvae. The gaping yolk sac larvae were predominantly colonized by members of the families Vibrionaceae and Flavobacteriaceae. Bacteria belonging to the Bacillus and Pseudoalteromonas genera were also present but less frequent. It was not possible to associate a type or group of bacteria directly related to "gaping". Strikingly, larvae with gaping jaws had an increase in the expression of two immune related genes, like hepcidin and chemokine (MIP-1B). These results indicate activation of the immune response in larvae with "gaping jaws" syndrome and this response could be related to bacteria isolated from gaping condition.

Ocean acidification and rising seawater temperature are environmental stressors resulting from the continuous increase of the atmospheric CO 2 concentration due to anthropogenic activities. As a consequence, marine fish are expected to undergo conditions outside of their tolerance range, leading to physiological challenges with possible detrimental implications. Our research group has previously shown that exposure to elevated CO 2 modulated the immune system of the Atlantic halibut. To further investigate this finding, we analysed non-specific immune components in blood plasma of Atlantic halibut ( Hippoglossus hippoglossus ) juveniles acclimated to six different temperatures (5, 10, 12, 14, 16 and 18 °C), and to water pH of 8.0 (control) or 7.6 (predicted for year 2100) for three months. Plasma ions (K + , Na + , Ca ++ , Cl − ) and lactate concentrations were also measured. The analysis of plasma ions did not show any trends related to temperature or CO 2 exposure, and the majority of the experimental fish were able to maintain ionic balance. The results show that both innate immune components (lysozyme and alternative complement system) had increased activities in response to elevated CO 2 , representing a CO 2 -related impact on the halibut's immune system. The increased activity of lysozyme and complement system is possibly part of the acclimatization process, and might be protective. [ABSTRACT FROM AUTHOR]

In the Northwest Atlantic, emerging evidence suggests that different stocks of Atlantic halibut (Hippoglossus hippoglossus) exist in Canadian and U.S. waters. To examine the movement of Atlantic halibut that occupy the Gulf of Maine in U.S. waters during the summer, two types of electronic tags were attached to large adult fish (n = 70) in 2007-2009. None of the recovered tags (n = 12) provided evidence that the fish occupied the relatively deep waters of the continental slope during the winter, where Canadian Atlantic halibut have been shown to spawn. This observation provides additional evidence for the hypothesis that different stocks of Atlantic halibut exist in Canadian and U.S. waters. Furthermore, this observation requires reexamination of the long-standing assumptions made by fisheries scientists that all Atlantic halibut spawn in deep water on the continental slope and that the majority of mature Atlantic halibut spawn annually. This information may be important for understanding the population dynamics of Atlantic halibut in U.S. waters. [ABSTRACT FROM AUTHOR]

One of the main problems facing Atlantic halibut hatcheries is the high mortality in the early stages of larval development. Several factors could be involved, for example: water quality, diseases or abnormalities, such as deformities occurring in the yolk sac larvae prior to exogenous feeding. The aim of this study was to identify differences in bacterial flora associated with yolk sac larvae with oral deformity. We also aimed to establish whether there is any relationship between bacterial strains and the "gaping jaws" syndrome. During our study, 74 bacterial isolates were obtained using three different nutrient media: Marine Agar, R2A and TCBS. Some of these bacteria were characterized using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and 16S rRNA sequencing. The immune response in larvae exhibiting the "gaping jaws" condition was measured by real time PCR. Our results showed significant differences in bacterial flora between normal and gaping larvae. The gaping yolk sac larvae were predominantly colonized by members of the families Vibrionaceae and Flavobacteriaceae. Bacteria belonging to the Bacillus and Pseudoalteromonas genera were also present but less frequent. It was not possible to associate a type or group of bacteria directly related to "gaping". Strikingly, larvae with gaping jaws had an increase in the expression of two immune related genes, like hepcidin and chemokine (MIP-1ß). These results indicate activation of the immune response in larvae with "gaping jaws" syndrome and this response could be related to bacteria isolated from gaping condition. [ABSTRACT FROM AUTHOR]

Background: Atlantic halibut (Hippoglossus hippoglossus) is a high-value, niche market species for cold-water marine aquaculture. Production of monosex female stocks is desirable in commercial production since females grow faster and mature later than males. Understanding the sex determination mechanism and developing sex-associated markers will shorten the time for the development of monosex female production, thus decreasing the costs of farming. Results: Halibut juveniles were masculinised with 17 α-methyldihydrotestosterone (MDHT) and grown to maturity. Progeny groups from four treated males were reared and sexed. Two of these groups (n = 26 and 70) consisted of only females, while the other two (n = 30 and 71) contained balanced sex ratios (50% and 48% females respectively). DNA from parents and offspring from the two mixed-sex families were used as a template for Restriction-site Associated DNA (RAD) sequencing. The 648 million raw reads produced 90,105 unique RAD-tags. A linkage map was constructed based on 5703 Single Nucleotide Polymorphism (SNP) markers and 7 microsatellites consisting of 24 linkage groups, which corresponds to the number of chromosome pairs in this species. A major sex determining locus was mapped to linkage group 13 in both families. Assays for 10 SNPs with significant association with phenotypic sex were tested in both population data and in 3 additional families. Using a variety of machine-learning algorithms 97% correct classification could be obtained with the 3% of errors being phenotypic males predicted to be females. Conclusion: Altogether our findings support the hypothesis that the Atlantic halibut has an XX/XY sex determination system. Assays are described for sex-associated DNA markers developed from the RAD sequencing analysis to fast track progeny testing and implement monosex female halibut production for an immediate improvement in productivity. These should also help to speed up the inclusion of neomales derived from many families to maintain a larger effective population size and ensure long-term improvement through selective breeding. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate a convenient, low-cost modification to conventional transfer methods for Atlantic halibut juveniles. A series of wire mesh cages were stacked within transport tanks creating a stratified transport system (STS), increasing the surface area for settlement and facilitating a more homogeneous distribution of halibut throughout the tank compared with the conventional insulated box (Unstructured, UTS). A stochastic cost-benefit analysis determined investment into a STS to be cost-effective, generating a mean benefit-cost ratio of 1.31 (95% CI, 0.68-2.00) after 2 years and a mean 5-year net present value of $85,176 (95% CI, $46,906-$125,630). The implementation of a STS was found to be technically feasible and economically efficient method to improve Atlantic halibut transport. [ABSTRACT FROM AUTHOR]

To evaluate the nutrient removal capabilities of two red macroalgae, apical blades were cultured in the lab for 4 weeks at either 6, 10, or 17°C and nitrate at either 30 or 300 μM, typical of the seasonal range of conditions at a land-based Atlantic halibut farm. Stocking density was 2.0 g L, irradiance 125 μmol photons m s, photoperiod 16:8 (L:D), and nitrogen to phosphorus ratio 10:1. For both species, the highest growth rate was at 300 μM NO with Palmaria palmata growing fastest at 6°C, 5.8% day, and Chondrus crispus growing best at 17°C, 5.5% day. Nitrogen and carbon removal by P. palmata was inversely related to temperature, the highest rate at 6°C and 300 μM NO of 0.47 mg N and 6.3 mg C per gram dry weight per day. In contrast, C. crispus removal of N was independent of temperature, with mean removal of 0.49 mgN gDW day at 300 μM NO. The highest carbon removal by C. crispus was 4.4 mgC gDW day at 10°C and 300 μM nitrate, though not significantly different from either 6 or 17°C and 300 μM nitrate. Tissue carbon:nitrogen ratios were >20 in both species at 30 μM nitrate, and all temperatures indicating nitrogen limitation in these treatments. Phycoerythrin content of P. palmata was independent of temperature, with means of 23.6 mg gFW at 300 μM nitrate. In C. crispus, phycoerythrin was different only between 6°C and 17°C at 300 μM nitrate, with the highest phycoerythrin content of 12.6 mg gFW at 17°C. Morphological changes were observed in P. palmata at high NO concentration as curling of the fronds, whilst C. crispus exhibited the formation of bladelets as an effect of high temperature. [ABSTRACT FROM AUTHOR]

In the present study, 10 polymorphic microsatellite DNA loci from Atlantic halibut (Hippoglossus hippoglossus) were isolated and characterized. The number of alleles for these loci ranged from 2 to 4 in tested 24 individuals. Observed and expected heterozygosities per locus varied from 0.21 to 0.70 and from 0.31 to 0.65, respectively. Most of these 10 microsatellite loci were successfully amplified and showed polymorphic in five related species. These loci will be useful for the assessment of genetic diversity and population structure of Atlantic halibut. [ABSTRACT FROM AUTHOR]

Abstract: The effects of photoperiod and feeding regimes on plasma IGF-I levels and their relationship with growth rate of juvenile halibut (initial mean weight 364 g) were investigated by rearing fish under five different photoperiod regimes and two feeding regimes for 14 months. The entire photoperiod experiment was divided into 3 phases where the fish in each phase were exposed to either natural photoperiod (N), stimulated photoperiod with long day and short night (S) or continuous light (L). Thus, the following five photoperiod combinations were tested: a) Control group (NNN) b) Group 2A (NLN) c) Group 2B (NNL) d) Long day-natural group (SNN) e) Production group (LNN). In addition, the Control group was split into two parts and fed according to two different feeding regimes: a) Continuous fed group: Fish fed every day. b) Starvation/re-fed group: Fish were starved for 5 weeks and then re-fed for 10 weeks, and the treatment repeated during the whole experimental period. The analyses of IGF-I were performed from individually tagged fish in all groups in September 2005 and March 2006. In order to test how rapidly starvation affects circulating IGF-I levels samples were taken from the Starvation/re-fed group after a 10 days starvation (September) and immediately after 10 weeks of feeding (March). A significant relationship between IGF-I levels and individual growth in the preceding period and photoperiod and starvation treatment was found on both occasions. In conclusion, the present study indicates that plasma IGF-I levels are correlated to growth in Atlantic halibut, and affected by photoperiod treatment or compensatory growth during re-feeding. Correlation between individual growth rate and IGF-I levels was low, but significant, highlighting the complexity of how environmental factors affect the endocrine and physiological regulation of growth in fish. [Copyright &y& Elsevier]

Abstract: Gene expression studies are fundamental to understand the molecular basis of severe malformations in fish development, particularly under aquaculture conditions. Real-time PCR (qPCR) is the most accurate method of quantifying gene expression, provided that suitable endogenous controls are used to normalize the data. To date, no reference genes have been validated for developmental gene expression studies in Atlantic halibut (Hippoglossus hippoglossus). We have determined the expression profiles of 6 candidate reference genes (Actb, Eef2, Fau, Gapdh, Tubb2 and 18S rRNA) in 6 embryonic and 5 larval stages of Atlantic halibut development. There were significant changes in expression levels throughout development, which stress the importance and complexity of finding appropriate reference genes. The three software applications (BestKeeper, geNorm and NormFinder) used to evaluate the stability of potential reference genes produced comparable results. Tubb2 and Actb were the most stable genes across the different developmental stages, whereas 18S rRNA and Gapdh were the most variable genes and thus inappropriate to use as reference genes. According to geNorm and NormFinder, the best two-gene normalization factors corresponded to the geometric average of Tubb2/Actb and Tbb2/Fau, respectively. We believe that either of these normalization factors can be used for future developmental gene expression studies in Atlantic halibut. [Copyright &y& Elsevier]

Abstract: Cataract pathology, intensity and severity, and temporal development were investigated in halibut in the unusual location for halibut of solely the anterior lens. This manifested as an opacity and there was fibre disruption in the anterior cortex with vacuoles. Individual eye blindness was 17–26% and significantly higher in non-migrated eyes, but bilateral blindness was only 0–4%. Cataract prevalence varied greatly in fish between sea pens, from 29 to 83%, and there was a significant increase on time, with a higher prevalence in migrated eyes. The cataract severity score per individual eye increased from 1.24 to 1.69 (score range 0–3) between sampling dates but there was no significant difference in severity in migrated and non-migrated eyes. Neither cataract prevalence nor severity were related to individual fish weight suggesting that growth per se was not an important influence. Physical damage largely affected the protruding non-migrated eye as a consequence of aggression in the hatchery and consequently physical damage of halibut in sea cages was well healed. Damage from UV light and nutritional deficiencies were regarded as possible factors in the formation of these anterior cataracts in ongrown halibut. [Copyright &y& Elsevier]

Abstract: Insulin-like growth factor-I (IGF-I) is an important regulator of growth and development in vertebrates. Both the endocrine and paracrine actions of IGF-I are mediated through ligand-binding to a membrane-bound IGF-I receptor (IGF-IR). The characterization of this receptor and subsequent expression studies thus help elucidate the endocrine regulation of developmental processes. As other flatfish species, the Atlantic halibut (Hippoglossus hippoglossus) undergoes a dramatic larval metamorphosis. This process is largely under endocrine control, and data indicate that IGF-I could be a key regulator. IGF-I content increases up to late pre-metamorphosis and decreases during metamorphosis. The IGF-IR has, however, not been studied during flatfish metamorphosis. To examine IGF-IR gene expression, two IGF-IR mRNA were cloned and sequenced. These partial sequences share high identity (≥95%) and similarity (≥97%) with other fish IGF-IR and lower identity (≥77%) and similarity (≥83.5%) with Japanese flounder insulin receptors. The expression of mRNA for both IGF-IR was analyzed by quantitative real-time RT-PCR during six larval developmental stages from pre- to post-metamorphosis. IGF-IR1 and IGF-IR2 mRNA are differentially expressed during metamorphosis, but if this indicates an isoform-specific regulation of developmental processes by circulating and/or locally-secreted IGF-I is unclear. Both IGF-IR genes are down-regulated in halibut larvae experiencing arrested metamorphosis, suggesting the IGF-I system is critical for metamorphic success in halibut. [Copyright &y& Elsevier]

Abstract: Continuous-elution electrophoresis (CEE) has been applied to separate putative hormones from adult Atlantic halibut pituitaries. Soluble proteins were separated by size and charge on Model 491 Prep Cell (Bio-Rad), where the homogenate runs through a cylindrical gel, and protein fractions are collected as they elute from the matrix. Protein fractions were assessed by SDS–PAGE and found to contain purified proteins of molecular size from 10 to 33kDa. Fractions containing proteins with molecular weights of approximately 21, 24, 28 and 32kDa, were identified as putative growth hormone (GH), prolactin, somatolactin and gonadotropins, respectively. These were analyzed further by mass spectrometry and identified with peptide mass protein fingerprinting. The CEE technique was used successfully for purification of halibut GH with a 5% yield, and appears generally well suited to purify species-specific proteins often needed for research in comparative endocrinology, including immunoassay work. Thus, the GH obtained was subsequently used as standards and iodination label in a homologous radioimmunoassay, applied to analyze GH content through larval development in normally and abnormally metamorphosing larvae. As GH is mainly found in the pituitary, GH contents were analyzed in tissue extracts from the heads only. The pituitary GH content increases proportionally to increased larval weight from first feeding to metamorphic climax. No difference in relative GH content was found between normal and abnormal larvae and it still remains to be established if GH has a direct role in metamorphosis. [Copyright &y& Elsevier]

An Atlantic Halibut (Hippoglossus hippoglossus) (41.4 cm fork length) with the same pigmentation on the blind side as on the ocular side (ambicoloration) was captured in the lower St. Lawrence estuary, Canada, on 24 June 2004. This specimen represents the most extreme case of ambicoloration published for this species to date. As in other cases of extreme ambicoloration, this specimen shows other signs of abridged metamorphosis and reduced asymmetry, such as incomplete migration of the left eye and a hooked dorsal fin. Studies of hatchery-reared larvae have shown that the most common cause of malpigmentation, including ambicoloration, in halibut is a diet containing inadequate amounts and proportions of essential unsaturated fatty acids shortly before metamorphosis. Even though not expected to be common in the wild, inadequate diet for a short duration before metamorphosis is the most likely cause of ambicolouration in wild halibut. [ABSTRACT FROM AUTHOR]

Fish larval development, not least the spectacular process of flatfish metamorphosis, appears to be under complex endocrine control, many aspects of which are still not fully elucidated. In order to obtain data on the functional development of two major endocrine glands, the pituitary and the thyroid, during flatfish metamorphosis, histology, immunohistochemistry and in situ hybridization techniques were applied on larvae of the Atlantic halibut ( Hippoglossus hippoglossus), a large, marine flatfish species, from hatching through metamorphosis. The material was obtained from a commercial hatchery. Larval age is defined as day-degrees (D°=accumulated daily temperature from hatching). Sporadic thyroid follicles are first detected in larvae at 142 D° (27 days post-hatch), prior to the completion of yolk sack absorption. Both the number and activity of the follicles increase markedly after yolk sack absorption and continue to do so during subsequent development. The larval triiodothyronine (T3) and thyroxine (T4) content increases, subsequent to yolk absorption, and coincides with the proliferation of thyroid follicles. A second increase of both T3 and T4 occurs around the start of metamorphosis and the T3 content further increases at the metamorphic climax. Overall, the T3 content is lower than T4. The pituitary gland can first be distinguished as a separate organ at the yolk sack stage. During subsequent development, the gland becomes more elongated and differentiates into neurohypophysis (NH), pars distalis (PD) and pars intermedia (PI). The first sporadic endocrine pituitary cells are observed at the yolk sack stage, somatotrophs (growth hormone producing cells) and somatolactotrophs (somatolactin producing cells) are first observed at 121 D° (23 days post-hatch), and lactotrophs (prolactin producing cells) at 134 D° (25 days post-hatch). Scarce thyrotrophs are evident after detection of the first thyroid follicles (142 D°), but coincident with a phase in which follicle number and activity increase (260 D°). The somatotrophs are clustered in the medium ventral region of the PD, lactotrophs in the anterior part of the PD and somatolactotrophs are scattered in the mid and posterior region of the pituitary. At around 600 D°, coinciding with the start of metamorphosis, somatolactotrophs are restricted to the interdigitating tissue of the NH. During larval development, the pituitary endocrine cells become more numerous. The present data on thyroid development support the notion that thyroid hormones may play a significant role in Atlantic halibut metamorphosis. The time of appearance and the subsequent proliferation of pituitary somatotrophs, lactotrophs, somatolactotrophs and thyrotrophs indicate at which stages of larval development and metamorphosis these endocrine cells may start to play active regulatory roles. [ABSTRACT FROM AUTHOR]

Viral haemorrhagic septicaemia (VHS) is a well-characterized disease of rainbow trout, Oncorhynchus mykiss, which has also caused economic losses in marine turbot farms in the British Isles. We have previously demonstrated that turbot, Scophthalmus maximus, are susceptible to isolates of viral haemorrhagic septicaemia virus (VHSV) that are endemic in the marine environment, highlighting a potential risk to marine aquaculture. Given the increasing interest in the intensive rearing of additional aquaculture species such as Atlantic cod, Gadus morhua, and Atlantic halibut, Hippoglossus hippoglossus, this study aimed at investigating the susceptibility of these species to VHSV. Both species were found to be largely resistant to VHS following immersion challenge with a selection of 18 isolates, representing the known marine VHSV genotypes. Only one and two VHSV-associated mortalities occurred out of a total of 1710 and 1254 halibut and cod, respectively. These findings suggest that there is a low direct risk to the development of commercial cod and halibut aquaculture from the existing endemic reservoir of VHSV. This study, coupled to field observations has, however, highlighted the fact that both species can become infected with VHSV. The known adaptability of RNA viruses, together with the selection pressures associated with intensive aquaculture would thus advocate a cautious approach to VHSV surveillance within these emerging industries. [ABSTRACT FROM AUTHOR]

Abstract Atlantic halibut, Hippoglossus hippoglossus (L.), was shown to be sensitive to infection by three different isolates of Aeromonas salmonicida ssp. achromogenes in pre-challenge tests using intraperitoneal (i.p.) and intramuscular (i.m.) injections as well as bath challenges. A commercial furunculosis vaccine, Alphaject 1200, and an autogenous vaccine, AAS, based on the challenge strain, induced immune protection as shown in challenge tests 8 weeks post-immunization. The survival rate of vaccinated fish after i.p. challenge was 100%, whereas mortality of control fish was 61%. Employing i.m. challenge, relative percentage survival induced by the furunculosis vaccine and the AAS vaccine was 47 and 44, respectively. Mortality of i.m. injected controls was 68%. Vaccinated fish behaved normally following vaccination but the weight gain was significantly reduced in vaccinated fish 8 weeks post-vaccination compared with control fish receiving phosphate-buffered saline. At the same time, intra-abdominal adhesions were observed in fish injected with either of the two vaccines or adjuvant alone. Antibody response against A. salmonicida ssp. achromogenes was detected in sera from fish receiving either vaccine. [ABSTRACT FROM AUTHOR]

For many aquaculture finfish species, the current broodstock have been collected from the wild or have undergone only a few generations of domestication. The Atlantic halibut (Hippoglossus hippoglossus) aquaculture industry in Atlantic Canada has retained F1 juveniles (n=145) from the 1996 spawning of wild adults for candidate broodstock. Through the development and use of a five-microsatellite DNA marker multiplex, we determined the parentage of these 1996 F1 individuals, which are being reared at one government and two industry hatcheries, and evaluated the change in genetic variation between the wild and the 1996 F1 stock. In the three groups of F1 fish, single parental pairs were assigned to 98%, 96% and 100% of individuals. Large full- and half-sibling groups were identified within and across F1 groups and, overall, only 36% of attempted crosses were represented in the retained fish. Effective population size in the parental group decreased from 27 to 13 when variance in family size was accounted for and to 12.5 when changes in gene diversity (compared to the combined F1 stocks) were considered. Statistically significant differences in measures of genetic variation were not widely observed between groups (original wild sample, parental group, three F1 groups and combined F1). However, the F1 population shows a 26% decrease in total allele numbers compared to the wild sample. These observations demonstrate the utility of genetic tools in the evaluation of genetic diversity and determination of pedigree during the establishment of new broodstock. They also emphasize the necessity for closely monitoring future matings among these fish and suggest the need to introduce additional genetic variation into this group of Atlantic halibut broodstock. [Copyright &y& Elsevier]

The assessment of the acceptable level of fish farm development in Scottish coastal areas is strongly dependent on the predicted rate of release of nitrogenous nutrients and particulate organic waste. A mass balance model of 4-week resolution has been used to estimate the rate of production of dissolved and particulate waste by cultivated halibut (Hippoglossus hippoglossus L.). The predicted release of dissolved nitrogen is 48 kg N/t of fish produced and that of particulate organic waste is 150–200 kg/t production. Modelled biomass-specific rates varied within a range of a factor of about three during the growing cycle, averaging 8.3 mg of dissolved N/kg biomass of fish/h and 28.8 mg/kg/h of faeces. There is experimental evidence to indicate potential for future developments to lead to significant improvements in feed utilisation by halibut in cages and an accompanying reduction in waste output. Comparisons with experimental data highlight areas where discrepancies between model and measured values indicate that further studies are required. Research targets include the partitioning of dissolved nitrogen between ammonia, urea and other soluble compounds, and the effects of growth rate and feed utilisation efficiency on waste production. [Copyright &y& Elsevier]

The bacterial flora of reared Atlantic halibut eggs, larvae, juveniles and adults from three different UK halibut hatcheries was monitored using a combination of traditional biochemical tests, the BIOLOG GN bacterial identification system, PCR-RFLP of 16S rRNA genes and partial 16S rDNA gene analysis.A gut microflora was detected towards the beginning of the non-feeding yolk-sac stage, with a substantial increase as the larvae started to feed. Bacteria isolated from non-feeding yolk-sac larvae were predominantly non-fermentative Gram-negative rods, in particular Pseudoalteromonas species; by contrast, the presumptive gut microflora of first-feeding larvae was generally dominated by members of the Vibrio genus.There was evidence of a bacterial succession after the larvae started to feed. Initially, first-feeding halibut reared on enriched Artemia were generally colonised by live food-associated bacteria, particularly Vibrio splendidus and V. alginolyticus-type organisms. Genetically similar V. splendidus isolates were recovered from enriched Artemia and the guts of first-feeding halibut larvae in all three hatcheries. However, only a selected proportion of the Artemia-associated bacteria appeared able to establish themselves within the guts of larval halibut.First-feeding halibut larvae were sometimes colonised by opportunistic bacteria. The gut floras of two individual batches of halibut larvae were briefly dominated by a Pseudoalteromonas and a Photobacterium phosphoreum isolate, respectively.Genetically-similar V. salmonicida-type organisms were present in larvae sampled from all three hatcheries, both from copepod and Artemia-fed fish. The guts of the adult halibut analysed had a culturable flora dominated by Photo. phosphoreum. [Copyright &y& Elsevier]

Atlantic halibut (Hippoglossus hippoglossus L.) females grow larger and mature later than males, making monosex (all-female) culture economically advantageous. The objective of this research was to develop methods for the production of monosex populations of halibut through the use of sex steroids. Six hundred postlarval halibut, mean size 30.0 mm fork length (FL), were divided evenly among eight tanks and fed a formulated diet supplemented with either 17α-methyldihydrotestosterone (MDHT; 1 and 5 ppm) or 17β-estradiol (E2; 10 ppm) for 45 days. Fish were then further reared on a standard halibut diet until sex ratios could be determined through histological and macroscopic observations. The MDHT treatments yielded 97–100% males, while the E2 treatment yielded a 70–74% female population. This demonstrates that the direct feminization of halibut can be achieved, and also allows for the evaluation of indirect feminization through the crossing of hormonally masculinized females (“neomales”) to normal females. [Copyright &y& Elsevier]

The aim of the study was to compare theantioxidant systems in juvenile marine fish ofcommercial importance to European aquaculture,namely turbot ( Scophthalmus maximus),halibut ( Hippoglossus hippoglossus) andgilthead sea bream ( Sparus aurata). Thepresent dietary trial was specifically designedto investigate the antioxidant effects ofvitamin E under moderate oxidising conditions,including high dietary levels of highlyunsaturated fatty acids and the feeding ofoxidised oils. The objective was to induce astressful pro-oxidant status to enablecharacterisation of the biochemical responsesto peroxidative stress without causingunnecessary suffering to the experimentalanimals or high mortalities during the trials. Both sea bream and turbot showed excellentgrowth, whereas growth was poorer in halibut.Dietary oxidised oil significantly reducedgrowth in turbot and especially in halibut, butnot in sea bream. Vitamin E improved growth insea bream fed oxidised oil but not in turbot orhalibut. However, vitamin E supplementationappeared to improve survival in all threespecies. In sea bream and turbot, liverantioxidant defence enzyme activities weregenerally increased by feeding peroxidised oiland reduced by vitamin E. Conversely, inhalibut, the liver antioxidant defence enzymeactivities were not increased by feedingperoxidised oil and only superoxide dismutasewas reduced by feeding vitamin E. Consistentwith these data, feeding oxidised oil increasedlipid peroxidation products in halibut, butgenerally not in sea bream or turbot.Furthermore, lipid peroxidation products weregenerally reduced by dietary vitamin E in bothsea bream and turbot, but not in halibut. Therefore, halibut liver antioxidant defenceenzymes did not respond to dietary oxidised oilor vitamin E as occurred in turbot and,especially sea bream. This resulted inincreased levels of lipid peroxides in halibutcompared to turbot and sea bream in fish givendietary oxidised oil. In addition, supplementalvitamin E did not reduce lipid peroxides inhalibut as it did in turbot and sea bream. Theincreased peroxidation stress in halibut mayaccount for their poorer growth and survival incomparison to turbot and especially sea bream.Halibut were reared at a lower temperature,although relatively high for halibut, thaneither turbot or sea bream but they were alsoslightly younger/smaller fish and possibly,therefore, more developmentally immature, andeither or all of these factors may be importantin the lack of response of the liver enzymes inhalibut. [ABSTRACT FROM AUTHOR]

At 7 days after first feeding (DAFF), the peptide hormone cholecystokinin (CCK) content (fmol individual−1) and the tryptic activity [μmol arginine-methyl-coumarinyl-7-amide (MCA) min−1 individual−1] per individual gut of Atlantic halibut Hippoglossus hippoglossus larvae were low: 0·2±0·1 and 0·14±0·10, respectively. Thereafter, both parameters increased with the increase in gut mass and reached 19·67±5·58 and 2·71±0·64 at 26 DAFF, respectively. Due to the small sample size, the dry mass (MG, mg) of the individual gut could not be determined accurately at 7 DAFF. At 13 DAFFMG represented 5·5% of whole body dry mass (MW, mg) while at 26 DAFF it had increased to 23%. The mass specific tryptic activity [μmol MCA min−1 per mg dry mass (M)] in the gut increased from 2·74±1·98 at 13 DAFF to 5·00±0·78 at 26 DAFF. There was more individual variation in the mass specific CCK content (fmol M−1) but no significant differences were found, although the data indicated an increase (from 23·38±11·26 at 13 DAFF to 36·27±8·96 fmol M−1 at 26 DAFF). At 7 DAFF the CCK content of the gut represented c. 2% of the whole body CCK content while it increased to c. 62% of the whole body CCK content at 26 DAFF. This demonstrates that it is necessary to separate neural and gastrointestinal sources of CCK in order to determine its alimentary role in fish larvae. Trypsin activity was only found in the gut compartment. In larvae aged 45 DAFF dietary proteins delivery into the gut by tube-feeding appeared to stimulate post-prandial secretion of CCK from the gut as well as stimulate pancreatic trypsin secretion, suggesting that both factors contribute to protein digestion. Copyright 2002 The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved. [Copyright &y& Elsevier]

In order to enhance growth, survival and quality during early juvenile stages of marine fish it is important to avoid lipid oxidation problems that are known to cause pathologies and disease. The aim of the present study was to characterize and compare the antioxidant systems in juvenile marine fish of commercial importance in European aquaculture, namely turbot (Scophthalmus maximus ), halibut (Hippoglossus hippoglossus ) and gilthead sea bream (Sparus aurata ). The experiment investigated the interaction of the dietary antioxidant micronutrient, vitamin E, with antioxidant defence systems. Fish were fed with diets of identical unsaturation index supplemented with graded amounts of vitamin E. The relationships between dietary and subsequent tissue vitamin E levels were determined as well as the effects of vitamin E supplementation on lipid and fatty acid compositions of both liver and whole fish, on the activities of the liver antioxidant defence enzymes, and on the levels of liver and whole body lipid peroxidation products, malondialdehyde (thiobarbituric acid reactive substances, TBARS) and isoprostanes. Growth and survival was only significantly affected in sea bream where feeding the diet with the lowest vitamin E resulted in decreased survival and growth. A gradation was observed in tissue vitamin E and polyunsaturated fatty acid (PUFA)/vitamin E levels in response to dietary vitamin E levels in all species. The activities of the main radical scavenging enzymes in the liver, catalase, superoxide dismutase and glutathione peroxidase generally reflected dietary and tissue vitamin E levels being highest in fish fed with the lowest level of vitamin E. The indicators of lipid peroxidation gave consistent results in all three species, generally being highest in fish fed with the unsupplemented diet and generally lowest in fish fed with the diet with highest vitamin E. In this respect, isoprostane levels generally paralleled TBARS levels supporting their value as... [ABSTRACT FROM AUTHOR]

The process of gonadal sex differentiation in 338 Atlantic halibut Hippoglossus hippoglossus larvae, ranging in size from 10 mm LS to 230 mm LF, is described histologically. Gonadal sex differentiation occurred by 38·0 mm LF, which coincided with the weaned, post-metamorphic, settled stage in the life cycle. This was a gradual process that coincided with other organogenesis in the developing larvae. Copyright 2002 The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved. [Copyright &y& Elsevier]

Considering that amino acids constitute an important energy fuel during early life of the Atlantic halibut (Hippoglossus hippoglossus L.), it is of interest to understand how the nitrogenous end products are handled. In this study we focused on the kinetics and fates of ammonia, urea and uric acid. The results showed that ammonia (TAmm: NH3+NH4+), and urea–N contents increased during final oocyte maturation. Urea–N excretion dominated the total nitrogenous end product formation in early embryos. Later, yolk TAmm levels increased in embryos and ammonia excretion was low. In the last part of the embryonic stage TAmm accumulation dominated, and was apparently due to yolk storage. Around hatching, the larval body tissues (larva with yolk-sac removed) accounted for 68% of whole animal urea–N accumulation, while TAmm levels increased predominately by yolk accumulation. Afterwards, ammonia excretion dominated and uric acid accumulation accounted for less than 1%. Urea, synthesised either through the ornithine–urea cycle, argininolysis or uricolysis, accounted for approximately 8% of total nitrogenous end product formation in yolk-sac larvae. The results suggested that a sequence occurred regarding which nitrogenous end products dominated and how they were handled. Urea excretion dominated in early embryos (<7 dPF), followed by yolk ammonia accumulation (7–12 dPF), and finally, ammonia excretion dominated in later embryonic and yolk-sac larval stages (>12 dPF). [Copyright &y& Elsevier]

Growth of juvenile Atlantic halibut from three areas of the North Atlantic (Canada, Iceland and Norway) was studied in an experiment using individual tagged fish reared at 15°C for 85 days. Fish from each population were subsequently split into two groups and acclimatised to either 12°C or 18°C. The fish were then injected intra-peritoneally with a Vibrio anguillarum bacteria suspension and mortality monitored for 4 weeks. Growth rates of the Canadian population ranked lowest, whereas the Norwegian population had the highest mean growth rates (SGR=1.70% day−1, 1.62% day−1 and 1.53% day−1 for the Norwegian, Icelandic and Canadian populations, respectively). The halibut from Norway had the best survival following bacterial challenge (80%, 50% and 55% survival for the Norwegian, Icelandic and Canadian populations, respectively). Mortality was higher at 18°C than at 12°C in the Icelandic (62% at 12°C and 27% at 18°C) and Canadian (56% at 12°C and 32% at 18°C) fish, whereas a smaller difference between temperatures was observed in the Norwegian fish (25% at 12°C and 13% at 18°C). Fish that survived the challenge test were those that had grown fastest in the growth trial. Low, but significant, correlations between survival and size and growth were seen, but these correlations varied between populations. In the Canadian population, no correlation between size and growth and survival were seen; only size was correlated (r=0.27) with survival in the Icelandic population, whereas both size (r=0.18) and growth (r=0.17) were correlated with survival in the Norwegian population. [Copyright &y& Elsevier]

Theaim of this study was to determine whether it is possible to influence thespecies composition of the gut microflora in 70-days old halibut larvae( Hippoglossus hippoglossus L.) by addition of bacteriabioencapsulated in Artemia franciscana in two shortpulses.Two Vibrio strains, PB 1-11 and PB 6-1 were used. Twotreatments received each strain alone, a third treatment received the twobacterial strains in mixture, whereas in a control treatment no bacteria wereadded. Five fish tanks were included in each treatment. The bacteria werebioencapsulated in Artemia franciscana metanauplii duringashort-term incubation in bacterial suspensions of the specific strains, andwereadded in two pulses, on day 1 and day 10 of the experiment. Addition ofbacteriadid not increase the total number of colony-forming units (CFU) in the larvalgut. The total CFU in the water was lower in tanks added bioencapsulatedbacteria than in the control treatment ( p < 0.05). Animmunocolony blot assay was used to measure the numbers of the specificbacteriaadded in samples of water, A. franciscana, and fishlarvae.At the end of the experiment on day 13, the strain PB 1-11 reached on average10% of the total CFU when added alone and 25% when added in mixture with PB6-1.Strain PB 6-1 reached at the end of the experiment 26.8% of the total CFU whenadded alone and 24.2% when added in mixture with PB 1-11, whereas thepercentages of PB 1-11 and PB 6-1 in the control fish were 12.7% and 10.8% ofthe total CFU, respectively. However, the differences between the experimentaltreatments compared with the control group were not significant( p > 0.05). The addition of bioencapsulated bacteria intwo pulses within a ten days period was therefore not sufficient to influencethe species composition of the microflora of the halibut larvae. [ABSTRACT FROM AUTHOR]

Total ammonia content (TAmm) and excretion (JAmm), and ionic content (K+, Na+, Cl-) have been studied in embryos and yolk-sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.) maintained in darkness (6 °C and 34% S) or in light (light-arrested, nonon-hatching embryos). The TAmm of the eggs increased from 250-300 nmol ind-1 during the first 5-10 days post fertilization (dPF) to 400-500 nmol ind-1 at the time of natural hatching in darkness. TAmm did not start to decrease before 2-5 days after hatch, and yolk-sac larvae had a higher TAmm compared to the light-arrested embryos of the same age and batch. JAmm increased exponentially rom about 0.05 nmol ind-1 h-1 at 10 dPF to about 3 nmol ind-1 h-1 at hatch. Embryos undergoing hatching showed a significantly higher JAmm than non-hatching, light-arrested embryos of the same age. The content of K+ in distilled water rinsed eggs declined from about 0.85 µmol ind-1 at 2-4 dPF to about 0.15 µmol ind-1 at hatch, while that of Cl- remained constant at about t 0.85 µmol in-1, and that of Na+ increased slowly from 0.10 to 0.20 µmol ind-1 . Based on the measured ions, there was an anionic deficit in the egg, especially during the first week of development. When exposed to increased ambient total ammonia (0-27 mM NH4Cl), no mortality occurred, and no significant increase was found in the TAmm of the Atlantic halibut egg before 9.2 dPF despite an estimated large inward diffusion gradient for the un-ionized ammonia species (NH3). The delayed release of TAmm comp ared to embryos and yolk-sac larvae of other marine teleosts, may relate to the deep-water spawning of the Atlantic halibut, and the buoyancy regulation of the yolk-sac larvae. [ABSTRACT FROM AUTHOR]

CO 2 emissions from human activities are increasing, resulting in greater rates of change in the oceans, exceeding any other event in geological and historical records over the past 300 million years. Oceans are warming and pH levels are decreasing. Marine organisms will need to respond to multiple stressors and the potential consequences of global change-related effects in fish needs to be investigated. Fish are affected by many biotic and abiotic environmental variables, including temperature and CO 2 fluctuations, and it is therefore critical to investigate how these variables may affect physiological and biochemical processes. We investigated the effects of elevated CO 2 levels (pH of 8.0, which served as a control, or 7.6, which is predicted for the year 2100) combined with exposure to different temperatures (5, 10, 12, 14, 16, and 18 °C) in the Atlantic halibut (Hippoglossus hippoglossus) during a three month experiment. Since regulation of reactive oxygen species (ROS) is crucial for physiological processes the focus was on the antioxidant defense system and we assessed the effects on catalytic activities of antioxidant enzymes (SOD, CAT, GR, GST, GPx). In addition we also analyzed effects on cholinesterase enzymes (AChE and BChE), and CYP1A enzyme activities (EROD). The treatments resulted in oxidative stress, and damage was evident in the form of protein carbonyls which were consistently higher in the elevated CO 2 -treated fish at all temperatures. Analyses of antioxidant enzymes did not show the same results, suggesting that the exposure to elevated CO 2 increased ROS formation but not defences. The antioxidant defense system was insufficient, and the resulting oxidative damage could impact physiological function of the halibut on a cellular level. Unlabelled Image [ABSTRACT FROM AUTHOR]