Tora Bardal

Molecular ontogenesis of digestive capability and associated endocrine control in Atlantic cod (Gadus morhua) larvae.

We have profiled the expression of twelve genes, in order to provide an overview on the molecular ontogeny of digestive capability with the associated endocrine control during Atlantic cod (Gadus morhua) larval development. Enzyme activity levels for the key digestive enzyme, trypsin, was also measured. Specifically, transcripts for trypsin, amylase, lipolytic enzymes: bile salt activated lipase (BAL), phospholipase A2 (PLA2) and Acyl CoA dehydrogenase (ACADM), regulatory peptides: neuropeptide Y (NPY), orexin (OX) cholecystokinin (CCK) and cocaine and amphetamine-related transcript (CART), the somatotropic factors: growth hormone (GH), preprosomatostatin 1 (PPSS1) and thyroid hormone receptors (TRα and TRβ) were analyzed using quatitative (real-time) polymerase chain reaction (qPCR). Trypsin and BAL mRNA levels peaked at approximately day 17 and 25 post-hatch, respectively, and thereafter displayed a decreasing pattern until metamorphosis. GH mRNA levels decreased moderately from 3 to 33dph, and thereafter, an increase was observed until 46dph. TRα mRNA levels showed a fluctuating pattern peaking at day 39 post-hatch. TRβ mRNA levels were too low to obtain quantitative measurements. Amylase mRNA slightly increased from day 3 to 17 post-hatch, and thereafter showed a steady decrease until day 60. Interestingly, PLA2 mRNA expression showed a consistent increase throughout the study period, indicating an increasingly important role during larval development. Overall, data from this study indicate that cod larvae show differential developmental mode of expression patterns for key genes and endocrine factors that regulate digestive capability, growth and development. These data are discussed in relation to larval trypsin enzyme activity and previous reports for other teleost species.

Somite formation and expression of MyoD, myogenin and myosin in Atlantic halibut (Hippoglossus hippoglossus L.) embryos incubated at different temperatures: transient asymmetric expression of MyoD.

SUMMARY Genes encoding the myogenic regulating factors MyoD and myogenin and the structural muscle proteins myosin light chain 2 (MyLC2) and myosin heavy chain (MyHC) were isolated from juvenile Atlantic halibut (Hippoglossus hippoglossus L.). The impact of temperature on their temporal and spatial expression during somitogenesis were examined by incubating halibut embryos at 4, 6 and 8°C, and regularly sampling for whole-mount in situ hybridisation and reverse transcription (RT)–PCR. There were no significant effects of temperature on the onset of somitogenesis or number of somites at hatching. The rate of somite formation increased with increasing temperature, and the expression of MyoD, myogenin and MyHC followed the cranial-to-caudal somite formation. Hence, no significant effect of temperature on the spatial and temporal expression of the genes studied was found in relation to somite stage. MyoD, which has subsequently been shown to encode the MyoD2 isoform, displayed a novel bilaterally asymmetric expression pattern only in white muscle precursor cells during early halibut somitogenesis. The expression of myogenin resembled that previously described for other fish species, and preceded the MyHC expression by approximately five somites. Two MyLC2 cDNA sequences were for the first time described for a flatfish, probably representing embryonic (MyLC2a) and larval/juvenile (MyLC2b) isoforms. Factors regulating muscle determination, differentiation and development have so far mostly been studied in vertebrates with external bilateral symmetry. The findings of the present study suggest that more such investigations of flatfish species could provide valuable information on how muscle-regulating mechanisms work in species with different anatomical, physiological and ecological traits.

Effects of n-3 and n-6 Fatty Acids on Tumor Necrosis Factor Cytotoxicity in WEHI Fibrosarcoma Cells

Modulation by fatty acids of the cytotoxic effect of recombinant tumor necrosis factor alpha (TNF) toward WEHI 164 mouse fibrosarcoma cells has been examined. Preincubating the highly TNF-sensitive WEHI clone 13 cells for 44 hr with 50 μmol/L of 20∶5n−3, 22∶6n−3, 18∶3n−6, 20∶3n−6 or 20∶4n−6 reduced cell survival 22 hr after challenge with TNF (40 ng/L) by 65%, 72%, 60%, 98% and 85%, respectively. In comparison, 18∶3n−3, 18∶2n−6 and 18∶1n−9 had only negligible effects on TNF-induced toxicity. Different extent of fatty acid incorporation into cell total phospholipids or triglycerides could not explain the observed effects on TNF cytotoxicity, and the enhanced cytotoxicity could therefore not be explained merely by an increased unsaturation of the cell membranes. In addition to the fatty acid supplied, preincubation with 18∶2n−6, 18∶3n−6 or 18∶3n−3 also enriched the cells with 20∶2n−6, 20∶3n−6 and 20∶3n−3, respectively, most likely due to chain elongation. The results suggest that the WEHI cells have a low Δ6 desaturase activity, and that n−6 and n−3 acids must have at least 3 or 4 double bonds, respectively, to enhance TNF cytotoxicity in WEHI cells. Dexamethasone partly inhibited TNF-induced cytotoxicity, while cyclooxygenase, thromboxane synthetase or lipoxygenase inhibitors had no or negligible effects. The antioxidant butylated hydroxyanisole (BHA) completely inhibited TNF-induced cytotoxicity, while the structurally and functionally similar antioxidant butylated hydroxy-toluene had no such effect, indicating that BHA does not block TNF cytotoxicity through its antioxidant effect. The results suggest that TNF cytotoxicity involves, directly or indirectly, metabolism of long-chain polyun-saturated fatty acids, and we speculate that fatty acid metabolites are involved.

Molecular characterization of a PDZ–LIM protein in Atlantic salmon (Salmo salar): a fish ortholog of the α-actinin-associated LIM-protein (ALP)

A protein containing both PDZ and LIM protein–protein interaction motifs has for the first time been identified in a lower vertebrate species. A full-length cDNA encoding the ortholog of the α-actinin-associated LIM protein (ALP) was isolated from white skeletal muscle of Atlantic salmon (Salmo salar). Whereas ALP is expressed as two muscle specific isoforms in mammals and chicken as the result of alternative splicing, a single ALP transcript was found in both muscle and non-muscular tissues of Atlantic salmon. On the other hand, Western blot analysis revealed several immunoreactive ALP variants in salmon muscle tissues, including a 45 kDa protein in white and red skeletal muscle and a 37–40 kDa protein in heart and smooth muscle. Salmon ALP and α-actinin showed similar striated patterns in serial longitudinal sections of white and red skeletal muscle and heart muscle. Expression of ALP was initiated at the 45-somite stage of the salmon embryogenesis contemporary with the first appearance of α-actinin transcripts. The similarities in both the spatial and temporal expression patterns of salmon ALP and α-actinin strongly indicate that the two proteins are associated as in higher vertebrates, and that the assumed involvement of ALP in the organization and/or maintenance of the Z-lines in striated muscle has been conserved during vertebrate evolution. However, in contrast to the restricted expression of ALP in higher vertebrates, the ubiquitous expression of salmon ALP suggest that this factor is involved in the assembly of additional multi-protein complexes in fish.

Molecular cloning of the Atlantic salmon activin receptor IIB cDNA – Localization of the receptor and myostatin in vivo and in vitro in muscle cells

In mammals, the activin receptor type IIB (ActRIIB) binds with high affinity several members of the transforming growth factor-beta (TGF-beta) superfamily, including the negative muscle regulator myostatin (MSTN). In this study, an actRIIB cDNA of 1443 bp was isolated by reverse transcription (RT)-PCR from the liver of Atlantic salmon (Salmo salar) encoding almost the complete receptor. The deduced salmon ActRIIB of 481 amino acids (aa) contained the conserved catalytic domain of serine/threonine protein kinases, and showed the highest sequence identity (83-87%) to the zebrafish, chicken and goldfish ActRIIB. Salmon actRIIB mRNA was identified by RT-PCR in all the examined tissues of juvenile fish that was confirmed by in situ hybridization. In comparison, the salmon MSTN signal was less widespread, and co-expression of the receptor and this putative ligand was only demonstrated in skeletal muscle. Consistently, both ActRIIB and MSTN were immunocytologically identified in salmon myoblasts and differentiated myotubes in culture.

Differential spatio-temporal expression and functional diversification of the myogenic regulatory factors MyoD1 and MyoD2 in Atlantic halibut (Hippoglossus hippoglossus)

Development of the vertebrate skeletal muscle is orchestrated by the myogenic regulatory factors MyoD, Myf5, myogenin and MRF4, which likely arose from the duplications of a single ancestral gene early in vertebrate evolution. We have isolated two myod genes from Atlantic halibut and examined their differential expression during embryogenesis using quantitative PCR and in situ hybridization to address their functional roles in this asymmetrically organized flatfish. myod1 was initially maternally expressed, while myod2 mRNA was first detectable during gastrulation. The myod1 mRNA levels predominated throughout somitogenesis, and both slow and fast muscle precursor cells displayed the bilateral symmetric myod1 signal during the formation of the symmetric somite pairs. In contrast, myod2 was left-right asymmetrically expressed in the fast muscle precursors. The random expression of myod2 was not associated with the right-sided eye migration and the development of thicker fast skeletal muscle on the eyed side than on the blind side. The nucleotide substitution analysis indicated that the teleost MyoDs essentially have evolved under purifying selection, but a subset of amino acid sites under strong positive selection were identified in the MyoD2 branch. Altogether, halibut MyoD1 seems to have retained the central role of MyoD in driving skeletal myogenesis, whereas the function of MyoD2 is uncertain in this flatfish species.

An oil containing EPA and DHA from transgenic Camelina sativa to replace marine fish oil in feeds for Atlantic salmon (Salmo salar L.): Effects on intestinal transcriptome, histology, tissue fatty acid profiles and plasma biochemistry

New de novo sources of omega 3 (n-3) long chain polyunsaturated fatty acids (LC-PUFA) are required as alternatives to fish oil in aquafeeds in order to maintain adequate levels of the beneficial fatty acids, eicosapentaenoic and docosahexaenoic (EPA and DHA, respectively). The present study investigated the use of an EPA+DHA oil derived from transgenic Camelina sativa in Atlantic salmon (Salmo salar) feeds containing low levels of fishmeal (35%) and fish oil (10%), reflecting current commercial formulations, to determine the impacts on tissue fatty acid profile, intestinal transcriptome, and health of farmed salmon. Post-smolt Atlantic salmon were fed for 12-weeks with one of three experimental diets containing either a blend of fish oil/rapeseed oil (FO), wild-type camelina oil (WCO) or transgenic camelina oil (DCO) as added lipid source. The DCO diet did not affect any of the fish performance or health parameters studied. Analyses of the mid and hindgut transcriptomes showed only mild effects on metabolism. Flesh of fish fed the DCO diet accumulated almost double the amount of n-3 LC-PUFA than fish fed the FO or WCO diets, indicating that these oils from transgenic oilseeds offer the opportunity to increase the n-3 LC-PUFA in farmed fish to levels comparable to those found a decade ago.

Quality of Atlantic Cod Frozen in Cell Alive System, Air-Blast, and Cold Storage Freezers

ABSTRACT Gutted Atlantic cod, packed in cartons, were frozen immediately after killing in a magnetic field (cell alive system). The results were compared with traditional air-blast freezing or by putting the cartons directly in a cold storage room (without forced convection of air). After frozen storage, external and fillet properties were compared. In spite of differences in freezing rates, only minor differences were found among treatments. The mechanism for the freezing of fish in the magnetic field, under the current conditions, appeared to be similar to that of traditional freezing methods.

Allometric growth and development of organs in ballan wrasse (Labrus bergylta Ascanius, 1767) larvae in relation to different live prey diets and growth rates

ABSTRACT Small fish larvae grow allometrically, but little is known about how this growth pattern may be affected by different growth rates and early diet quality. The present study investigates how different growth rates, caused by start-feeding with copepods or rotifers the first 30 days post-hatch (dph), affect allometric growth and development of nine major organs in ballan wrasse (Labrus bergylta) larvae up to experimental end at 60 dph. Feeding with cultivated copepod nauplii led to both increased larval somatic growth and faster development and growth of organ systems than feeding with rotifers. Of the organs studied, the digestive and respiratory organs increased the most in size between 4 and 8 dph, having a daily specific growth rate (SGR) between 30 and 40% in larvae fed copepods compared with 20% or less for rotifer-fed larvae. Muscle growth was prioritised from flexion stage and onwards, with a daily SGR close to 30% between 21 and 33 dph regardless of treatment. All larvae demonstrated a positive linear correlation between larval standard length (SL) and increase in total tissue volume, and no difference in allometric growth pattern was found between the larval treatments. A change from positive allometric to isometric growth was observed at a SL close to 6.0 mm, a sign associated with the start of metamorphosis. This was also where the larvae reached postflexion stage, and was accompanied by a change in growth pattern for most of the major organ systems. The first sign of a developing hepatopancreas was, however, first observed in the largest larva (17.4 mm SL, 55 dph), indicating that the metamorphosis in ballan wrasse is a gradual process lasting from 6.0 to at least 15-17 mm SL. Summary: Allometric growth patterns of ballan wrasse were not affected by different diets, and larval functional development was found to be dependent on size and not age or growth rate.

Skeletal muscle growth dynamics and the influence of first-feeding diet in atlantic cod larvae (Gadus morhua L.)

ABSTRACT Dynamics between hypertrophy (increase in cell size) and hyperplasia (increase in cell numbers) of white and red muscle in relation to body size [standard length (SL)], and the influence of the first-feeding diets on muscle growth were investigated in Atlantic cod larvae (Gadus morhua). Cod larvae were fed copepod nauplii or rotifers of different nutritional qualities from 4 to 29 days post hatching (dph), Artemia nauplii from 20 to 40 dph and a formulated diet from 36 to 60 dph. The short period of feeding with cultivated copepod nauplii had a positive effect on both muscle hyperplasia and hypertrophy after the copepod/rotifer phase (19 dph), and a positive long term effect on muscle hypertrophy (60 dph). The different nutritional qualities of rotifers did not significantly affect muscle growth. We suggest here a model of the dynamics between hyperplasia and hypertrophy of red and white muscle fibre cells in relation to cod SL (4 to 30 mm), where the different red and white muscle growth phases clearly coincided with different metamorphosis stages in cod larvae. These shifts could be included as biomarkers for the different stages of development during metamorphosis. The main dietary muscle effect was that hypertrophic growth of red muscle fibres was stronger in cod larvae that were fed copepods than in larvae that were fed rotifers, both in relation to larval age and size. Red muscle fibres are directly involved in larval locomotory performance, but may also play an important role in the larval myogenesis. This can have a long term effect on growth potential and fish performance. Summary: Hyperplastic and hypertrophic growth dynamics of red and white muscle were strongly related to cod larval size and corresponded with the metamorphosis process. First-feeding diet quality can prolong effects on muscle growth potential in cod larvae.