Antifreeze protein dispersion in eelpouts and related fishes reveals migration and climate alteration within the last 20 Ma.

Antifreeze proteins inhibit ice growth and are crucial for the survival of supercooled fish living in icy seawater. Of the four antifreeze protein types found in fishes, the globular type III from eelpouts is the one restricted to a single infraorder (Zoarcales), which is the only clade know to have antifreeze protein-producing species at both poles. Our analysis of over 60 unique antifreeze protein gene sequences from several Zoarcales species indicates this gene family arose around 18 Ma ago, in the Northern Hemisphere, supporting recent data suggesting that the Arctic Seas were ice-laden earlier than originally thought. The Antarctic was subject to widespread glaciation over 30 Ma and the Notothenioid fishes that produce an unrelated antifreeze glycoprotein extensively exploited the adjoining seas. We show that species from one Zoarcales family only encroached on this niche in the last few Ma, entering an environment already dominated by ice-resistant fishes, long after the onset of glaciation. As eelpouts are one of the dominant benthic fish groups of the deep ocean, they likely migrated from the north to Antarctica via the cold depths, losing all but the fully active isoform gene along the way. In contrast, northern species have retained both the fully active (QAE) and partially active (SP) isoforms for at least 15 Ma, which suggests that the combination of isoforms is functionally advantageous.

Delayed phenotypic expression of growth hormone transgenesis during early ontogeny in Atlantic salmon (Salmo salar)?

Should growth hormone (GH) transgenic Atlantic salmon escape, there may be the potential for ecological and genetic impacts on wild populations. This study compared the developmental rate and respiratory metabolism of GH transgenic and non-transgenic full sibling Atlantic salmon during early ontogeny; a life history period of intense selection that may provide critical insight into the fitness consequences of escaped transgenics. Transgenesis did not affect the routine oxygen consumption of eyed embryos, newly hatched larvae or first-feeding juveniles. Moreover, the timing of early life history events was similar, with transgenic fish hatching less than one day earlier, on average, than their non-transgenic siblings. As the start of exogenous feeding neared, however, transgenic fish were somewhat developmentally behind, having more unused yolk and being slightly smaller than their non-transgenic siblings. Although such differences were found between transgenic and non-transgenic siblings, family differences were more important in explaining phenotypic variation. These findings suggest that biologically significant differences in fitness-related traits between GH transgenic and non-transgenic Atlantic salmon were less than family differences during the earliest life stages. The implications of these results are discussed in light of the ecological risk assessment of genetically modified animals.

Helical antifreeze proteins have independently evolved in fishes on four occasions.

Alanine-rich α-helical (type I) antifreeze proteins (AFPs) are produced by a variety of fish species from three different orders to protect against freezing in icy seawater. Interspersed amongst and within these orders are fishes making AFPs that are completely different in both sequence and structure. The origin of this variety of types I, II, III and antifreeze glycoproteins (AFGPs) has been attributed to adaptation following sea-level glaciations that occurred after the divergence of most of the extant families of fish. The presence of similar types of AFPs in distantly related fishes has been ascribed to lateral gene transfer in the case of the structurally complex globular type II lectin-like AFPs and to convergent evolution for the AFGPs, which consist of a well-conserved tripeptide repeat. In this paper, we examine the genesis of the type I AFPs, which are intermediate in complexity. These predominantly α-helical peptides share many features, such as putative capping structures, Ala-richness and amphipathic character. We have added to the type I repertoire by cloning additional sequences from sculpin and have found that the similarities between the type I AFPs of the four distinct groups of fishes are not borne out at the nucleotide level. Both the non-coding sequences and the codon usage patterns are strikingly different. We propose that these AFPs arose via convergence from different progenitor helices with a weak affinity for ice and that their similarity is dictated by the propensity of specific amino acids to form helices and to align water on one side of the helix into an ice-like pattern.

Epithelial dominant expression of antifreeze proteins in cunner suggests recent entry into a high freeze-risk ecozone.

Most marine teleost fishes residing in a high freeze-risk ecozone, such as the coastal waters of Newfoundland during winter, avoid freezing by secreting high concentrations of antifreeze proteins (AFP) into their blood plasma where they can bind to and prevent the growth of ice that enter the fish. Cunner (Tautogolabrus adspersus), which overwinter in such shallow waters are the only known exception. Although this species does produce type I AFP, the plasma levels are too low to be of value as a freeze protectant. Southern and Northern blot analyses carried out in this study establish that the cunner AFP genes belong to a multigene family that is predominantly expressed in external epithelia (skin and gill filaments). These results support the hypothesis that the survival of cunner in icy waters is attributable in part to epithelial AFP that help block ice propagation into their interior milieu. In contrast to the cunner, heterospecifics occupying the same habitat have greater freeze protection because they produce AFP in the liver for export to the plasma as well as in external epithelia. Since the external epithelia would be the first tissue to come into contact with ice it is possible that one of the earliest steps involved in the evolution of freeze resistant fish could have been the expression of AFP in tissues such as the skin. We suggest that this epithelial-dominant AFP expression represents a primitive stage in AFP evolution and propose that cunner began to inhabit "freeze-risk ecozones" more recently than heterospecifics.

Antifreeze protein gene amplification facilitated niche exploitation and speciation in wolffish.

During winter, the coastal waters of Newfoundland can be considered a 'freeze risk ecozone' for teleost fishes, where the shallower habitats pose a high (and the deeper habitats a low) risk of freezing. Atlantic (Anarhichas lupus) and spotted (Anarhichas minor) wolffish, which inhabit these waters, reside at opposite ends of this ecozone, with the Atlantic wolffish being the species facing the greatest risk, because of its shallower niche. In order to resist freezing, this species secretes five times the level of antifreeze protein (AFP) activity into the plasma than does the spotted wolffish. The main basis for this interspecific difference in AFP levels is gene dosage, as the Atlantic wolffish has approximately three times as many AFP gene copies as the spotted wolffish. In addition, AFP transcript levels in liver (the primary source of circulating AFPs) are several times higher in the Atlantic wolffish. One explanation for the difference in gene dosage and transcript levels is the presence of tandemly arrayed repeats in the latter, which make up two-thirds of its AFP gene pool. Such repeats are not present in the spotted wolffish. The available evidence indicates that the two species diverged from a common ancestor at a time when the ebb and flow of northern glaciations would have resulted in the emergence of shallow water 'freeze risk ecozones'. The results of this study suggest that the duplication/amplification of AFP genes in a subpopulation of ancestral wolffish would have facilitated the exploitation of this high-risk habitat, resulting in the divergence and evolution of modern-day Atlantic and spotted wolffish species.

Thermolabile antifreeze protein produced in Escherichia coli for structural analysis.

The only hyperactive antifreeze protein (AFP) found to date in fishes is an extreme variant of the 3-kDa, alpha-helical, alanine-rich type I AFP, which is referred to here as type Ih. Purification of the 33-kDa homodimeric AFP Ih from a natural source was hampered by its low levels in fish plasma; by the need to remove the more abundant smaller isoforms; and by its extreme thermolability. Moreover, ice affinity as a purification tool was spoiled by the tendency of fish IgM antibodies to bind to ice in the presence of AFPs. In order to produce enough protein for crystallography we expressed AFP Ih as a recombinant protein in the Arctic Express® strain of Escherichia coli at 12 °C, just below the thermal denaturation temperature of 16-18 °C. His-tags were not useful because they compromised the activity and yield of AFP Ih. But in the absence of fish antibodies we were able to recover 10-mg quantities of the antifreeze protein using two cycles of ice affinity purification followed by anion-exchange chromatography to remove contaminating chaperones. The purified recombinant AFP Ih yielded diffraction-quality crystals with an extremely asymmetrical unit cell. By transferring the genes of the chaperones into a methionine auxotroph we were able to grow this host at low temperatures and produce sufficient selenomethionine-labeled AFP Ih for crystallography.

Isolation and characterization of type I antifreeze proteins from cunner, Tautogolabrus adspersus, order Perciformes.

Antifreeze proteins (AFPs) are produced by many species of teleost fish that inhabit potentially lethal ice-laden seawater and afford them protection from freezing. To date type I AFPs have been fully characterized in two teleost orders: Pleuronectiformes and Scorpaeniformes. In this study, we report the isolation and complete characterization of a type I AFP present in fish from a third order: cunner (Tautogolabrus adspersus), order Perciformes (family Labridae). This protein was purified from blood plasma and found to belong to what is now known as classical type I AFP with their small size (mass 4095.16 Da), alanine richness (> 57 mol%), high α-helicity (> 99%) with the ability to undergo reversible thermal denaturation, 11 amino acid (ThrX(10)) repeat regions within the primary structure, the capacity to impart a hexagonal bipyramidal shaping to ice crystals and the conservation of an ice-binding site found in many of the other type I AFPs. Partial de novo sequencing of the plasma AFP accounted for approximately half of the peptide mass. Sequencing of a combined liver and skin cDNA library indicated that the protein is produced without a signal sequence. In addition the translated product of the AFP cDNA suggests that it codes for the AFP isolated from plasma. These results further solidify the hypothesis that type I AFPs are multiphyletic in origin and suggest that they represent remarkable examples of convergent evolution within three orders of teleost fish.

A re-evaluation of the role of type IV antifreeze protein.

A lipoprotein-like antifreeze protein (type IV AFP) has previously been isolated only from the blood plasma of the longhorn sculpin. However, the plasma antifreeze activity in all individuals of this species tested from Newfoundland and New Brunswick waters ranges from low to undetectable. A close relative of the longhorn sculpin, the shorthorn sculpin, does have appreciable antifreeze activity in its blood but this is virtually all accounted for by the alpha-helical, alanine-rich type I AFP, other isoforms of which are also present in the skin of both fishes. We have characterized a putative ortholog of type IV AFP in shorthorn sculpin by cDNA cloning. This 12.2-kDa Gln-rich protein is 87% identical to the longhorn sculpin's type IV AFP. Recombinant versions of both orthologs were produced in bacteria and shown to have antifreeze activity. Immunoblotting with antibodies raised to type IV AFP shows this protein present in longhorn sculpin plasma at levels of less than 100 microg/mL, which are far too low to protect the blood from freezing at the temperature of icy seawater. This confirms the results of direct antifreeze assays on the plasmas. It appears that type IV AFP has the potential to develop as a functional antifreeze in these fishes but may not have been selected for this role because of the presence of type I AFP. Consistent with this hypothesis is the observation that the type IV AFP gene has not been amplified the way functional antifreeze protein genes have in all other species examined.

Antifreeze protein gene expression in winter flounder pre-hatch embryos: implications for cryopreservation.

Cryopreservation of fish embryos has proven to be an elusive goal. Two reasons for this lack of success are their high chilling sensitivity and the formation of ice crystals while in the frozen state or during the thawing process. Antifreeze proteins (AFP) that protect marine teleost fishes from freezing in subzero waters have been shown to be capable of inhibiting ice recrystallization and protecting cell membranes from cold induced damage. Therefore they have the potential to improve the success of embryo cryopreservation. A recent study demonstrated that vitrified winter flounder embryos continued to show developmental changes following thaw [V. Robles, E. Cabrita, G.L. Fletcher, M.A. Shears, M.J. King, M.P. Herráez, Vitrification assays with embryos from a cold tolerant sub-arctic fish species, Theriogenology 64 (2005) 1633-1646]. Since winter flounder produce AFP it was hypothesized that these proteins, if present in the embryos, could have contributed to this progressive step towards success. Winter flounder produce three species of type 1 AFP: a small liver type, a large "hyperactive" liver type and a skin type. This study was conducted to determine which, if any, of these AFP genes was being expressed in pre-hatch winter flounder embryos. There was no evidence of AFP activity in freshly fertilized embryos. However, low levels of AFP activity were found in embryos at 4, 8, and 11 days post-fertilization. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of the AFP mRNA isolated from the embryos revealed the expression of seven different skin type AFP genes that translated into four distinct AFP. Neither of the liver type AFP genes was expressed in the embryos.

Tissue specific expression of antifreeze protein and growth hormone transgenes driven by the ocean pout (Macrozoarces americanus) antifreeze protein OP5a gene promoter in Atlantic salmon (Salmo salar).

Previous research aimed at producing genetically improved salmon broodstock for aquaculture led to the creation of two lines of transgenic Atlantic salmon using gene constructs that were derived in part from the ocean pout OP5a antifreeze protein (AFP) gene. One of the lines was produced using an OP5a AFP gene in which the 5' region of the promoter was removed (termed t-OP5a-AFP), and the other line contains a growth hormone (GH) transgene (EO-1alpha) that consists of a chinook salmon GH cDNA driven by a truncated OP5a AFP promoter that is almost identical to that of the t-OP5a-AFP construct. The similarity of the promoter regions of these transgenes provided an opportunity to evaluate their tissue specific expression patterns. Expression of mRNA was evaluated using Northern blot and RT-PCR techniques. The results demonstrate that the AFP and GH trangenes were expressed in almost all body tissues, suggesting that the promoter region of the OP5a AFP gene lacks tissue specific elements. Northern analysis revealed that expression of the t-OP5a-AFP gene was considerably greater than that of the EO-1alpha GH transgene. Only the spleen tissue of the GH transgenics showed a visible band of hybridization. In contrast clear bands of hybridization were evident in all tissues, except for blood cells, of the AFP transgenics with heart, liver and brain tissue showing the highest levels of mRNA expression. This higher level of expression could be attributable to the presence of introns in the t-OP5a-AFP transgene. Since the GH transgenic salmon grow considerably faster than non-transgenics the low levels of GH transgene expression in this line were clearly sufficient to produce the desired rapid growth phenotype. In contrast the levels of AFP expression were inadequate to impart any improvement in the freeze resistance of the AFP transgenic salmon.

The importance of dissolved salts to the in vivo efficacy of antifreeze proteins.

Antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP) lower the freezing point of marine fish plasma non-colligatively by specifically adsorbing to certain surfaces of ice crystals, modifying their structure and inhibiting further growth. While the freezing point is lowered, the melting point is unaltered and the difference between the two is termed thermal hysteresis (TH). In pure water, the level of TH is directly related to the intrinsic activity of the specific AF(G)P in solution and to their concentration. Results of this study indicate that when AF(G)P are dissolved in salt solutions, such as NaCl, encompassing the range they could encounter in nature, there is a synergistic enhancement of basal TH that is positively related to the salt concentration. This enhancement is likely a result of the hydration shell surrounding the dissolved ions and, as a consequence, reducing freezable water. A secondary reason for the enhancement is that the salt could be influencing the hydration shell surrounding the AF(G)P, increasing their solubility and thus the protein surface area available to adsorb to the ice/water interface. The former hypothesis for the salt enhanced TH has implications for the in vivo function of AF(G)P, particularly at the seawater/external epithelia (gills, skin, stomach) interface. The latter hypothesis is likely only relevant to in vitro situations where freeze dried protein is dissolved in low salt solutions.

Characterization and multi-generational stability of the growth hormone transgene (EO-1alpha) 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-1alpha) 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-1alpha 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-1alpha 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-1alpha transgene codes for the chinook salmon GH, and that the transgene and the integration site have remained stable over multiple generations.

Type I antifreeze proteins expressed in snailfish skin are identical to their plasma counterparts.

Type I antifreeze proteins (AFPs) are usually small, Ala-rich alpha-helical polypeptides found in right-eyed flounders and certain species of sculpin. These proteins are divided into two distinct subclasses, liver type and skin type, which are encoded by separate gene families. Blood plasma from Atlantic (Liparis atlanticus) and dusky (Liparis gibbus) snailfish contain type I AFPs that are significantly larger than all previously described type I AFPs. In this study, full-length cDNA clones that encode snailfish type I AFPs expressed in skin tissues were generated using a combination of library screening and PCR-based methods. The skin clones, which lack both signal and pro-sequences, produce proteins that are identical to circulating plasma AFPs. Although all fish examined consistently express antifreeze mRNA in skin tissue, there is extreme individual variation in liver expression - an unusual phenomenon that has never been reported previously. Furthermore, genomic Southern blot analysis revealed that snailfish AFPs are products of multigene families that consist of up to 10 gene copies per genome. The 113-residue snailfish AFPs do not contain any obvious amino acid repeats or continuous hydrophobic face which typify the structure of most other type I AFPs. These structural differences might have implications for their ice-crystal binding properties. These results are the first to demonstrate a dual liver/skin role of identical type I AFP expression which may represent an evolutionary intermediate prior to divergence into distinct gene families.

Type I antifreeze proteins: possible origins from chorion and keratin genes in Atlantic snailfish.

Type I antifreeze proteins (AFPs) are alanine-rich alpha-helical polypeptides found in some species of right-eye flounders, sculpin, and snailfish. In this study, a shorthorn sculpin skin type I cDNA clone was used to probe an Atlantic snailfish liver cDNA library in order to locate expressed genes corresponding to snailfish plasma AFPs. Clones isolated from the cDNA library had sections with substantial amino acid and nucleotide sequence similarity to snailfish type I AFPs. However, further analysis revealed that the positives were actually three different liver-expressed proteins-two were eggshell proteins, while the third was a type II keratin. We propose that a shift in reading frame could produce alanine-rich candidate AFPs with possible antifreeze activity or ice crystal modification properties. Furthermore, it is plausible that one or more of the liver-expressed proteins represent the progenitors of snailfish type I AFPs.

Hyperactive antifreeze protein in flounder species. The sole freeze protectant in American plaice.

The recent discovery of a large hyperactive antifreeze protein in the blood plasma of winter flounder has helped explain why this fish does not freeze in icy seawater. The previously known, smaller and much less active type I antifreeze proteins cannot by themselves protect the flounder down to the freezing point of seawater. The relationship between the large and small antifreezes has yet to be established, but they do share alanine-richness (> 60%) and extensive alpha-helicity. Here we have examined two other righteye flounder species for the presence of the hyperactive antifreeze, which may have escaped prior detection because of its lability. Such a protein is indeed present in the yellowtail flounder judging by its size, amino acid composition and N-terminal sequence, along with the previously characterized type I antifreeze proteins. An ortholog is also present in American plaice based on the above criteria and its high specific antifreeze activity. This protein was purified and shown to be almost fully alpha-helical, highly asymmetrical, and susceptible to denaturation at room temperature. It is the only detectable antifreeze protein in the blood plasma of the American plaice. Because this species appears to lack the smaller type I antifreeze proteins, the latter may have evolved by descent from the larger antifreeze.

Isolation and purification of antifreeze proteins from skin tissues of snailfish, cunner and sea raven.

Antifreeze proteins/polypeptides (AFPs), which are found in diverse species of marine fish, are grouped into four distinct classes (types I-IV). The discovery of skin-specific type I AFPs established that this class contains distinct isoforms, liver-type and skin-type, which are encoded by separate gene families. In this study, type I AFPs were isolated and partially characterized from skin tissues of Atlantic snailfish (Liparis atlanticus) and cunner (Tautogolabrus adspersus). Interestingly, evidence from this study indicates that snailfish type I AFPs synthesized in skin tissues are identical to those circulating in their blood plasma. Furthermore, type II AFPs that are identical to those expressed in liver for export into blood were purified from sea raven (Hemitripterus americanus) skin tissue extracts. It is clear that epithelial tissues are an important source for antifreeze expression to enhance the complement of AFPs that protect fish from freezing in extreme cold environments. In addition, the evidence generated in this study demonstrates that expression of AFPs in fish skin is a widespread phenomenon that is not limited to type I proteins.

Hyperactive antifreeze protein in a fish.

Fish that live in the polar oceans survive at low temperatures by virtue of 'antifreeze' plasma proteins in the blood that bind to ice crystals and prevent these from growing. However, the antifreeze proteins isolated so far from the winter flounder (Pleuronectes americanus), a common fish in the Northern Hemisphere, are not sufficiently active to protect it from freezing in icy sea water. Here we describe a previously undiscovered antifreeze protein from this flounder that is extremely active (as effective as those found in insects) and which explains the resistance of this fish to freezing in polar and subpolar waters.

Spatial expression patterns of skin-type antifreeze protein in winter flounder (Pseudopleuronectes americanus) epidermis following metamorphosis.

Two isotypes of Type I antifreeze protein (AFP), the liver-type and the skin-type, have been described from adult winter flounder (Pseudopleuronectes americanus). Although the liver-type AFP has been well studied, the skin-type has just begun to be characterized. It appears to have a wide tissue distribution, be expressed constitutively, and the absence of a signal sequence suggests it is active intracellularly. The current study was designed to examine the onset of skin-type AFP expression during the thickening of the epidermis at metamorphosis from both the nucleic acid and protein levels. The epidermis appeared as a thin layer overlying a thickened dermis at metamorphosis and showed a gradual increase in thickness through the first fall and winter. The onset of skin-type antifreeze expression occurred in conjunction with this epidermal thickening. In situ hybridization and immunohistochemistry showed a distribution of mRNA and skin-type AFP specific for the epidermis and epidermal pavement cells. The AFP immunoproduct showed a distribution intimate with the pavement cell membrane and through the interstitial spaces. This distribution suggests that the AFP may be important in slowing ice crystal formation in these interstitial regions and thus reducing cellular damage due to osmotic imbalance.