Garth L. Fletcher

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.

Structural and Functional Similarity between Fish Antifreeze Proteins and Calcium-Dependent Lectins

A cDNA for a type II antifreeze protein was isolated from liver of smelt (Osmerus mordax). The predicted protein sequence is homologous to that from sea raven (Hemitripterus americanus) and both show homology to a family of calcium-dependent lectins. Smelt and sea raven belong to taxonomic orders believed to have diverged prior to Cenozoic glaciation. Thus, type II antifreeze proteins appear to have evolved independently in these fish species from pre-existing calcium-dependent lectins. Sequence alignment of the antifreezes and the lectins suggest that these proteins adopt a similar fold, that the sea raven antifreeze has lost its Ca2+ binding sites, and the smelt antifreeze has retained one site. Experiments show that smelt antifreeze protein activity is responsive to Ca2+ but that of sea raven antifreeze protein is not. These results suggest that the type II fish antifreeze proteins and calcium-dependent lectins share a common ancestry, related folding structures, and functional similarity.

TRANSGENIC FISH FOR AQUACULTURE

This review focuses on the technology currently being applied to produce transgenic fish, fish containing novel gene constructs that were experimentally introduced into their genome. A number of excellent overviews on this subject have been published in recent years (1–5). Thus rather than repeat what has already been well stated we have, where possible, attempted to examine critically the published results in order to determine the factors that have or have not been established as being important to the efficient production of stable lines of transgenic fish. We hope that this exercise will be useful to researchers currently applying, or contemplating applying, transgenic technology to fish, and that it will assist all of us in the design of experiments that will enable the production of transgenic fish to be as successful as the production of transgenic mice.

The potential impact of modern biotechnology on fish aquaculture

The introduction of molecular techniques in addition to the more traditional methods of biotechnology has supplied the resources to increase significantly production in world aquaculture. The ability to identify relevant genes endowing the phenotype of interest has certainly been helped by the ever-expanding databases, which have benefited not only from the various genome projects, but also from contemporary approaches such as the DNA chip, improved 2-D gel resolution and high throughput mass spectrometers. This, combined with improvements in transgenic technologies, has opened up vast possibilities to the aquacultural biotechnologist which include improving growth rates and cost-effectiveness, increasing resistance to pathogens and stressors, improving quality of broodstock and also creating the opportunity of making new or different products through altering their genetic make up. The platform technologies relevant to this field of functional genomics will be discussed in the context of applications beneficial to the field of aquaculture, while examples including those from our own research will be described.

Cardiorespiratory modifications, and limitations, in post-smolt growth hormone transgenic Atlantic salmon Salmo salar

SUMMARY In recent years, there has been a great deal of interest in how growth hormone (GH) transgenesis affects fish physiology. However, the results of these studies are often difficult to interpret because the transgenic and non-transgenic fish had very different environmental/rearing histories. This study used a stable line of size-matched GH Atlantic salmon (Salmo salar) that were reared in a shared tank with controls (at 10°C, for∼ 9 months) to perform a comprehensive examination of the cardiorespiratory physiology of GH transgenic salmon, and serves as a novel test of the theory of symmorphosis. The GH transgenic salmon had a 3.6× faster growth rate, and 21 and 25% higher values for mass-specific routine and standard oxygen consumption (ṀO2), respectively. However, there was no concurrent increase in their maximum ṀO2, which resulted in them having an 18% lower metabolic scope and a 9% reduction in critical swimming speed. This decreased metabolic capacity/performance was surprising given that the transgenics had a 29% larger heart with an 18% greater mass-specific maximum in situ cardiac output, a 14% greater post-stress blood haemoglobin concentration, 5–10% higher red muscle and heart aerobic enzyme (citrate synthase or cytochrome oxidase) activities, and twofold higher resting and 1.7× higher post-stress, catecholamine levels. However, gill surface area was the only cardiorespiratory parameter that was not enhanced, and our data suggest that gill oxygen transfer may have been limiting. Overall, this research: (1) shows that there are significant metabolic costs associated with GH transgenesis in this line of Atlantic salmon; (2) provides the first direct evidence that cardiac function is enhanced by GH transgenesis; (3) shows that a universal upregulation of post-smolt (adult) GH transgenic salmon cardiorespiratory physiology, as suggested by symmorphosis, does not occur; and (4) supports the idea that whereas differences in arterial oxygen transport (i.e. cardiac output and blood oxygen carrying capacity) are important determinants of inter-specific differences in aerobicity, diffusion-limited processes must be enhanced to achieve substantial intra-specific improvements in metabolic and swimming performance.

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

SummaryEight 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.

Lethal freezing temperatures of Arctic char and other salmonids in the presence of ice

Abstract The lethal freezing temperatures of seawater acclimated Arctic char (Salvelinus alpinus), brook trout (Salvelinus fontinalis), Atlantic salmon (Salmo salar), brown trout (Salmo trutta) and rainbow trout (Salmo gairdneri) were determined in the presence of ice. Arctic char showed the greatest resistance to freezing (−0.99°C) followed by brook (−0.87°C) and brown trout (−0.81°C) and by salmon (−0.76°C) and rainbow trout (−0.75°C). Plasma electrolyte levels accounted for 90–95% of the observed freezing temperature values. However, the lethal freezing temperatures of all of the salmonids were significantly lower than their plasma freezing temperatures, suggesting that at least one other factor was involved in preventing the fish from freezing. This difference between fish freezing temperature and plasma freezing temperature was greatest in Arctic char (0.2°C) and accounted for 70% of the difference in freezing resistance between char and salmon. It is hypothesized that the chars epidermis acts as a barrier to prevent ice propagation.

Winter Flounder Antifreeze Protein Improves the Cold Hardiness of Plant Tissues

Summary Exposure of plant tissues to the winter flounder antifreeze protein (AFP) has revealed three novel properties by which plant cold hardiness may be improved. Firstly, vacuum infiltration of the protein into leaves of potato, canola ( Brassica napus ) and Arabidopsis thaliana resulted in a significant depression of the spontaneous freezing temperature relative to water infiltrated controls. In the case of canola, the freezing temperature was decreased by an average of 1.8 °C. These results demonstrated the ability of the AFP to function as an anti-nucleator in plant tissues. Secondly, exposure of suspension cultured cells of bromegrass to the antifreeze protein resulted in a reduction in the amount of freezable water frozen at any given temperature. This showed that the protein could act as a cryoprotectant. Thirdly, the antifreeze protein decreased the rate of ice crystal formation. These results demonstrate the feasibility of improving the cold hardiness of plants by introduction of the antifreeze protein gene.

Liver-specific and seasonal expression of transgenic Atlantic salmon harboring the winter flounder antifreeze protein gene.

We have analyzed the inheritance and expression of a line of transgenic salmon harboring the antifreeze protein gene from the winter flounder. The genomic clone 2A-7 coding for a major liver-type antifreeze protein gene (wflAFP-6) was integrated into the salmon genome. From a transgenic founder (# 1469), an F3 generation was produced. In this study, southern blot analysis showed that only one copy of the antifreeze protein transgene was integrated into a unique site in F3 transgenic fish. The integration site was cloned and characterized. Northern analysis indicated that the antifreeze protein mRNA was only expressed in the liver and showed seasonal variation. All of the F3 offspring contained similar levels of the antifreeze protein precursor protein in the sera and the sera of these offspring showed a characteristic hexagonal ice crystal pattern indicating the presence of antifreeze activity. In addition, the antifreeze protein precursor protein level was found to vary with the season, being highest in the month of November and lowest in May. This study had demonstrated a tissue-specific and stable expression of the antifreeze protein transgene in the F3 generation of the transgenic salmon 1469 line.

The role of aquatic biotechnology in aquaculture

Abstract Food security will be a major issue facing mankind in the coming millennium. Aquacultural output will need to be increased several fold in order to meet the rising demands for fish in coming years. Biotechnology can provide the means to increase the intensity and capacity of the operation. Some of the platform technologies including DNA vaccines, DNA chips and proteomics and transgenic technology will be described. Transgenics, in particular, may make this farming process more efficient.