Antifreeze polypeptides from the Newfoundland ocean pout,Macrozoarces americanus: presence of multiple and compositionally diverse components

Summary Eight major antifreeze polypeptides (AFP) were purified from the sera of Newfoundland ocean pout. Except for their approximately identical size (6,000 Dalton), these components were shown to be separate entities by their behaviour on polyacrylamide gel electrophoresis, ion exchange chromatography, gel permeation and reverse phase high performance liquid chromatography. They could also be divided into two cross-reactive, yet distinct, immunological groups. Amino acid analysis demonstrated that ocean pout AFP are different from all of the other antifreezes studied to date. The ocean pout AFP do not contain the abundance of alanine (60 mol%) found in winter flounder and shorthorn sculpin AFP nor the high half-cystine residues (8 mol%) observed in sea raven AFP. It is suggested that ocean pout AFP represent a new type of macromolecular antifreeze.Abbreviations AFGP antifreeze glycoprotein(s) - AFP antifreeze polypeptide(s) - HPLC high performance liquid chromatography - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis     

The fish heart as a model system for the study of myoglobin

A model is presented for myoglobin study based upon naturally occurring differences in myocardial myoglobin content in fish. The sea raven (Hemitripterus americanus) and the ocean pout (Macrozoarces americanus) have heart myoglobin contents of approx. 65 and 5 nmol/g wet wt, respectively. The maximal activities of enzymes associated with energy metabolism are similar in the two hearts. Isolated perfused hearts performed with similar efficiencies based upon similar rates of work, oxygen consumption and lactate production. Under normoxic perfusion conditions both hearts met 98% of the ATP demand by oxidative mechanisms. Myoglobin-rich sea raven hearts performed significantly better than myoglobin-poor ocean pout hearts under conditions of hypoxia and glycolytic blockage. The performance of sea raven hearts was impaired during hypoxia by decreasing the content of functional myoglobin with hydroxylamine. No effect upon performance was observed with the ocean pout heart. The data provide the first evidence that myoglobin plays a role in the maintenance of contractility in heart under hypoxic conditions.     

Multiple genes provide the basis for antifreeze protein diversity and dosage in the ocean pout, Macrozoarces americanus

The ocean pout (Macrozoarces americanus) produces a set of antifreeze proteins that depresses the freezing point of its blood by binding to, and inhibiting the growth of, ice crystals. The amino acid sequences of all the major components of the ocean pout antifreeze proteins, including the immunologically distinct QAE component, have been derived by Edman degradation. In addition, sequences of several minor components were deduced from DNA sequencing of cDNA and genomic clones. Fifty percent of the amino acids are perfectly conserved in all these proteins as well as in two homologous sequences from the distantly related wolffish. Several of the conserved residues are threonines and asparagines, amino acids that have been implicated in ice binding in the structurally unrelated antifreeze protein of the righteye flounders. Aside from minor differences in post-translational modifications, heterogeneity in antifreeze protein components stems from amino acid differences encoded by multiple genes. Based on genomic Southern blots and library cloning statistics there are 150 copies of the 0.7-kilobase-long antifreeze protein gene in the Newfoundland ocean pout, the majority of which are closely linked but irregularly spaced. A more southerly population of ocean pout from New Brunswick in which the circulating antifreeze protein levels are considerably lower has approximately one-quater as many antifreeze protein genes. Thus, there appears to be a correlation between gene dosage and antifreeze protein levels, and hence the ability to survive in ice-laden seawater. Southern blot comparison of the two populations indicates that the differences in gene dosage were not generated by a simple set of deletions/duplications. They are more likely to be the result of differential amplification.     

Lack of metabolic thermal compensation during the early life stages of ocean pout Zoarces americanus (Bloch & Schneider) : a benthic, cold-water marine species

The present study investigated the metabolic response of young ocean pout Zoarces americanus to temperature acclimation (3 v. 11° C), and to acute changes in water temperature from 3 to 17° C. The Q ₁₀ value for standard metabolic rate between acclimation temperatures was 5·3, warm-acclimated fish displayed higher rates of oxygen uptake at all temperatures during the acute thermal challenge, and changes in whole-body citrate synthase activity were qualitatively similar to those seen for metabolism. These results indicate that, in contrast to temperate species, young ocean pout from Newfoundland do not show thermal compensation in response to long-term temperature changes.     

Structure of an antifreeze polypeptide and its precursor from the ocean pout, Macrozoarces americanus

Serum antifreeze polypeptides (AFP) from Newfoundland ocean pout have been resolved by ion exchange chromatography and reverse phase high performance liquid chromatography into at least 12 components. The protein sequences of three of the AFP were determined using a combination of protein Edman degradation and cDNA sequencing. The AFP precursor protein encodes for a preprotein of 87 amino acids with no obvious prosequences. Two of the AFP (SP1-A and SP1-C) were separate gene products with minor amino acid sequence differences. The protein structure of SP1-C precursor is MKSVILTGLLFVLLCVDHMTASQSVVAT QLIPINTALTPAMMEGKVTNPIGIPFAEMSQIVGKQVNTPVAKGQTLMPNMVKTYVAGK. The third AFP (SP1-B) is a post-translation modification product of SP1-C. These experiments indicate that the ocean pout AFP are a multigene family with protein structure different from any other known polypeptide antifreezes.     

Copulation, spawning and parental care in captive ocean pout

The ocean pout copulates through direct genital contact for internal fertilization. A complete spawning event consists of copulation, oviposition, and the female displaying parental care by wiping and wrapping herself around the eggs.     

Characterization and multi-generational stability of the growth hormone transgene (EO-1α) responsible for enhanced growth rates in Atlantic Salmon

Transgenic technologies provide a promising means by which desirable traits can be introduced into cultured fish species within a single generation thus accelerating the production of genetically superior broodstock for aquaculture. However, before such fish are allowed to be marketed as food they must receive government regulatory approval. Two pivotal regulatory requirements are: (1) complete characterization of the genomically integrated transgene and, (2) demonstration that the transgene remains stable over multiple generations. We have generated a stable line of growth hormone (GH) transgenic Atlantic salmon (Salmo salar) using an “all fish” gene construct (opAFP-GHc2) containing a growth hormone cDNA from chinook salmon whose expression is regulated by the 5′ promoter and 3′ termination regions derived from an ocean pout antifreeze protein (AFP) gene. In this study we show that a reorganized form of the opAFP-GHc2 construct (termed EO-1α) integrated as a single functional copy into a 35 bp repeat region of the genomic DNA. PCR based mapping revealed that the linear sequence of the EO-1α integrant was organized as follows: base pairs 1580–2193 of the ocean pout promoter region followed by the intact chinook salmon GH cDNA, the complete ocean pout antifreeze 3′ region, and the first 1678 bp of the ocean pout antifreeze 5′ region. Sequence analysis of the EO-1α integrant and genomic flanking regions in F₂ and F₄ generation salmon revealed that they were identical. In addition, apart from the disruption at the integration sites, the consensus sequences of the integrant in these two generations of salmon were identical to the sequence of the opAFP-GHc2 construct. These results indicate that the EO-1α transgene codes for the chinook salmon GH, and that the transgene and the integration site have remained stable over multiple generations.     

Myoglobin content and the activities of enzymes of energy metabolism in red and white fish hearts

Summary The myoglobin content of representative red and white coloured fish hearts was quantitated. It was confirmed that the macroscopic difference in appcarance is due to the presence or absence of myoglobin. Thereafter, the cytochrome c content as well as the maximal activities of key enzymes of energy metabolism were assessed in myoglobin-rich sea raven (Hemitripterus americanus) and myoglobin-poor ocean pout (Macrozoarces americanus) hearts. Both species are sluggish benthic dwellers that occur in similar habitats in the North Atlantic Ocean. The activities of enzymes associated with aerobic metabolism were similar in sea raven and ocean pout hearts, but far in excess of activities observed from white skeletal muscle. The two hearts also displayed comparable activities of enzymes associated with anaerobic energy metabolism. It therefore appears that the capacity to produce reducing equivalents for the electron transport system is similar in two selected fish hearts despite great differences in myoglobin content.     

Determination of the expression of fish antifreeze protein (AFP) in 7th generation transgenic mice tissues and serum

In this study, the presence of antifreeze protein (AFP) gene expression through successive generations in transgenic mice carrying the chimeric gene construct of the coding sequence for the AFP protein from ocean pout was investigated. AFP transgenic hemizygote mice were used for AFP gene expression. AFP genome expressions in transgenic mice were analyzed by Western blotting, and tissue location of AFP protein was shown by immunohistochemical and immunofluorescence techniques. Seventh transgenic mice from the established founders demonstrated the expression of AFP in organs such as the skin, oviduct, lung, kidney and liver tissues and serum except for the heart. Our results demonstrate successful expression of AFP gene products in several tissues and serum of transgenic mice, the association of in vivo expressed AFP protein, for the first time. These results indicate that the coding sequence for the AFP protein gene (ocean pout type III AFP gene) could be integrated and stably transcribed and expressed in the 7th generation of transgenic mice. In conclusion transgenic mouse lines would be a good model for the cryostudy of AFP and for the determination of AFP roles in several organs and tissues.     

Ultrastructure of the spermatozoa and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine fish

Ultrastructure of sperm and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine teleost, was examined by scanning and transmission electron microscopy. The results showed that the sperm do not have an acrosome but have a very long mid-piece (one to two times the sperm head length) containing numerous well-developed elongated mitochondria. The sperm also have two tails (is biflagellate) each consisting of nine peripheral and one central pair (9 ± 2) of microtubules. This long mid-piece and the biflagellate nature of the sperm appear to be associated with the long life-span of the sperm and with sperm dispersal in the ovary to fertilize the eggs internally. The ocean pout eggs are enveloped by a porous chorionic membrane similar to that found in other teleosts but have two micropyles, a condition likely related to a mechanism of egg fertilization which increases the egg fertlity in the presence of low sperm numbers. Following insemination, some biochemically undefined excretions appeared on the surface of fertilized eggs and led to the acquisition of adherent capability of the eggs which formed a tightly associated egg mass in sea water. © 1995 wiley-Liss, Inc.     

Development of an all-fish gene cassette for gene transfer in aquaculture.

To develop an all-fish gene cassette suitable for gene transfer in aquaculture, the antifreeze protein (AFP) gene promoter from the ocean pout (Macrozoarces americanus) was analyzed for its ability to direct exogenous gene expression both in vitro and in vivo. The ocean pout AFP (opAFP) gene promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) was functionally analyzed in two fish cell lines and in Japanese medaka embryos. The opAFP gene promoter was active in these systems, as demonstrated by the transient expression of CAT activity. These results suggest that the opAFP gene promoter is useful for many other gene transfer experiments. To facilitate use of the opAFP gene promoter as a common and versatile vehicle for fish gene transfers, an expression vector, opAFP-V, was constructed by linking the 2.1-kb opAFP gene promoter, the 63-bp opAFP gene 5' untranslated sequence, and the 1.2-kb opAFP gene 3' sequence by two unique restriction sites, Bg/II and HpaI, respectively. Thus, genes of interest can be inserted into either the Bg/II site or the HpaI site depending on the length of their 5' untranslated sequence. The complete DNA sequence of opAFP-V was determined to facilitate future detailed analysis of integration and expression of the transgene.     

Sperm Mediated Transfer of Genes into Goldfish and Detection by Polymerase Chain Reaction

Goldfish sperms were mixed with eggs for fertilization after incubation with antifreeze protein gene(AFP)from ocean pout for 30 min.A number of embryos and 145 adult goldfish were obtained.DNA from adult goldfish and embryos was extracted separately.Results of the amplification by PCRand Southern blot molecular hybridization indicate the integration of exogenous antifreeze gene intothe genome of a part of the recipient goldfish.Of the 45 samples detected by PCR,twelve showedpositive reaction with distinct hybridization band.The positive rate was 26%.     

Expression of a Novel Piscine Growth Hormone Gene Results in Growth Enhancement in Transgenic Tilapia (Oreochromis Niloticus)

Several lines of transgenic G1 and G2 tilapia fish (Oreochromis niloticus) have been produced following egg injection with gene constructs carrying growth hormone coding sequences of fish origin. Using a construct in which an ocean pout antifreeze promoter drives a chinook salmon growth hormone gene, dramatic growth enhancement has been demonstrated, in which the mean weight of the 7 month old G2 transgenic fish is more than three fold that of their non transgenic siblings. Somewhat surprisingly G1 fish transgenic for a construct consisting of a sockeye salmon metallothionein promoter spliced to a sockeye salmon growth hormone gene exhibited no growth enhancement, although salmon transgenic for this construct do show greatly enhanced growth. The growth enhanced transgenic lines were also strongly positive in a radio-immuno assay for the specific hormone in their serum, whereas the non growth enhanced lines were negative. Attempts to induce expression from the metallo thionein promoter by exposing fish to increased levels of zinc were also unsuccessful.Homozygous transgenic fish have been produced from the ocean pout antifreeze/chinook salmon GH construct and preliminary trials suggest that their growth performance is similar to that of the hemizygous transgenics. No abnormalities were apparent in the growth enhanced fish, although minor changes to skull shape and reduced fertility were noted in some fish. There is also preliminary evidence for improved food conversion ratios when growth enhanced transgenic tilapia are compared to their non-transgenic siblings.The long term objective of this study is to produce lines of tilapia which are both growth enhanced and sterile, so offering improved strains of this important food fish for aquaculture.     

The distribution of 0-group flatfish in relation to abiotic factors on the tidal flats in the brackish Dollard (Ems Estuary, Wadden Sea)

The distribution of 0-group flatfish was investigated in 1992 in the Dollard (Ems–Dollard estuary, Wadden Sea). 0-Group plaice, flounder and sole were not evenly distributed over the sampled locations. The spatial distribution pattern of 0-group flatfish in the Dollard changed during the investigation period. In the first week of sole presence, when the mean length of sole was 24–30 mm, salinity correlated significantly with sole density. The distribution of juvenile sole larger than 40 mm total length was affected by the elevation of the location: 0-group sole was restricted to the sampled site with the lowest elevation. The distribution of 0-group plaice was related to sediment: no juvenile plaice were caught at locations with more than 10% mud fraction in the sediment. The distribution of 0-group flounder was also correlated with sediment. Later in the year, salinity correlated negatively with the distribution of 0-group flounder. The influence of sediment composition is probably indirect and linked to the abundance of preferred food items, such as Corophium volutator . Abiotic conditions were suitable to 0-group plaice, flounder and sole.     

Ecological significance of 0-group fish in the Barents Sea ecosystem

Investigations into the 0-group fish in the Barents Sea have been carried out since 1965, with the goal of estimating the abundance of 0-group fish. 0-group abundance indices have been used in the assessment of the recruitment level and in recruitment variability studies. However, the ecological importance of the 0-group fish in the Barents Sea has been less studied. Although 0-group capelin, herring, cod and haddock are widely distributed in the Barents Sea, the central area seems to be the most important, accounting for approximately 50–80% of the annual biomass. The total biomass of the four most abundant 0-group fish species can be up to 3.3 million tonnes, with an average of 1.3 million tonnes (1993–2009). Wide distribution and high biomass of pelagically distributed 0-group fish make these fishes an important element in the energy transport between different trophic levels and different geographical areas, having a critical impact on the entire Barents Sea ecosystem. In recent years, capelin have shown a pronounced northward shift in biomass distribution, and several successive strong year classes occurred during warm temperature conditions. Cod biomasses were unexpectedly low during warm years and were positively correlated with spawning stock biomass, while the correlation with temperature was not significant. Haddock and herring show, as expected, increasing biomass with increased temperature when the spawning stock is at a sufficiently high level.     

Abundance,structure, growth and origin of inshore clupeid populations of the west coast of Scotland

The abundance, structure, growth, and origin of clupeid populations in inshore waters of the west coast of Scotland were studied from April 1970 to October 1972. Clupeid populations in the area comprise mainly young fish. 0-group autumn-spawned herring, probably of Minch origin, move into the area about April and spring-spawned ones (Clyde origin) about June. The timing and the body length at which each group arrives in the area during the different years is the same. After the initial immigration, the distribution of both 0-group clupeids becomes localized.Herring populations in the sea-lochs and associated inshore waters are mainly 0-group fish, which are replaced each year by a new incoming brood. The sprat populations of the sea-lochs are dominated by the 0-group; in the more ‘open’ areas the populations comprise older individuals. Year-class distribution in the ‘open’ areas resemble that of the commercial fishery.The rate of increase of 0-group autumn-spawned herring is 3.68 mm/week and that of spring-spawned 0-group herring is 2.83 mm/week. The curves of growth of 0-group sprats of the 1970 and 1971 year-classes are different; the rate of increase in length, however, averages 3.55 mm/week for both year-classes and the differences are not significant.In sprats, after their first year of life a rapid increase in length takes place in the spring. This increase is thought to enable the majority of the population to reach the minimum size (88–90 mm) for initial gonadal maturation and thereby make them capable of reproducing in their second year of life.     

Effect of glycosylation on hydration behavior at the ice-binding surface of the Ocean Pout type III antifreeze protein: a molecular dynamics simulation

Antifreeze proteins (AFPs), found in certain vertebrates, plants, fungi and bacteria have the ability to permit their survival in subzero environments by thermal hysteresis mechanism. However, the exact mechanism of ice growth inhibition is still not clearly understood. Here, four long explicit molecular dynamics (MD) simulations have been carried out at two different temperatures (277 and 298 K) with and without glycan to study the conformational rigidity of the Ocean pout type III antifreeze protein in aqueous medium and the structural arrangements of water molecules hydrating its ice-binding surface. It is found that irrespective of the temperature the ice-binding surface (IBS) of the protein is relatively more rigid than its non ice-binding surface (NonIBS) in its native and glycosylated form. Hydrophilic residues N14, T18 and Q44 are essential to antifreeze activity. Radial distribution, density distribution function and nearest neighbor orientation plots with respect to individual two surfaces confirm that density of water molecule near these binding surface in native and glycosylated form are relatively more than the nonbinding surface. The glycosylated form shows a strong peak than the native one. From rotational auto correlation function of water molecules around ice-binding sites, it is prominent that with increase in temperature, strong interaction between the water oxygen and the hydrogen bond acceptor group on the protein-binding surface decreases. This provides a possible molecular reason behind the ice-binding activity of ocean pout at the prism plane of ice.     

Distribution and diet of 0-group cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>) and haddock (<Emphasis Type="Italic">Melanogrammus aeglefinus</Emphasis>) in the Barents Sea in relation to food availability and temperature

Distribution of 0-group cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) in August–September 2005 and 2006 was mainly restricted to the Atlantic waters of the western and central areas of the Barents Sea. The main distribution of 0-group fish overlapped largely with areas of high biomass (>7 gm⁻² dry weight) of zooplankton. The copepod Calanus finmarchicus and krill Thysanoessa inermis, which are dominant zooplankton species in both Atlantic and boreal waters of the Barents Sea, were the main prey of 0-group cod and haddock. The main distribution, feeding areas and prey of 0-group cod and haddock overlapped, implying that competition for food may occur between the two species. However, though their diet coincided to a certain degree, haddock seems to prefer smaller and less mobile prey, such as Limacina and appendicularians. As 0-group fish increased in size, there seems to be a shift in diet, from small copepods and towards larger prey such as krill and fish. Overall, a largely pelagic feeding behaviour of 0-group cod and haddock was evident from this study.     

Expression and characterization of an active and thermally more stable recombinant antifreeze polypeptide from ocean pout, Macrozoarces americanus, in Escherichia coli: improved expression by the modification of the secondary structure of the mRNA.

The cDNA clone coding for the ocean pout antifreeze polypeptide (AFP) was modified to improve translation of its mRNA in Escherichia coli. A recombinant AFP (rAFP), MetLys-AFP-Lys, was expressed successfully using the lambda PL promoter, and constituted 1-2% of total bacterial proteins. The rAFP was purified to homogeneity from the soluble fractions of bacterial extracts. Its identity was confirmed by amino acid analysis, automated Edman degradation, immuno-blot and activity measurements. Although the rAFP is indistinguishable from the authentic AFP in its secondary structure, thermal hysteretic activity and the alteration of ice crystal structure, it is, however, thermally more stable (approximately 4.5 degrees C increase in Tm) and is more effective in inhibiting ice growth along the a-axis. These investigations indicate that the extra amino acids in rAFP significantly improve the thermal stability and ice-binding activity of the polypeptide.