The idea of an “antifreeze” system was first described in marine fish inhabiting the coast of Northern Labrador whose body fluids had the same freezing point of seawater (−1.7 °C to −2 °C) rather than freshwater (0 °C) (Scholander and others 1957; Gordon and others 1962). This phenomenon was eventually attributed to a set of peptides and glycopeptides termed AFPs and antifreeze glycoproteins (AFGPs), respectively. These proteins are synthesized primarily in the liver and secreted into the blood and extracellular space, where they bind and modify the structure of microice crystals, thereby inhibiting ice crystal growth and lowering the freezing point of body fluids (Davies and Hew 1990; Raymond 1991). AFPs have diverse structures and are divided into 3 categories (types I, II, and III) depending on their protein sequences. Also, the number of copies and type of AFP genes varies with the fish species: for example, winter flounder (Pleuronectes americanus) have 30 to 40 copies of type I; sea raven (Hemitripterus americanus) have 12 to 15 copies of type II; and Newfoundland ocean pout have 150 copies of type III (Hew and others 1995). 

The ocean pout type III AFP transgene has been successfully transferred and expressed in goldfish (Wang and others 1995). The gene was microinjected into the goldfish oocytes and was inherited through 2 generations. The transgenic goldfish showed significantly higher cold tolerance as compared to controls, suggesting possible use of the transgene for promoting cold resistance in fish. 

AFP transgenic technology could be highly beneficial to the aquaculture industry in countries with freezing and subzero coastline conditions. For example, winter water temperatures along the Atlantic coast of Canada can range from 0 °C to −1.8 °C; these conditions restrict the cultivation of salmonids and other commercially important fish to a few select areas at the southern edge of eastern Canada (Hew and others 1995; Fletcher and others 2004). Therefore, research is currently under way to develop strains of Atlantic salmon that could be cultivated over a wider geographic range. This could be accomplished by (1) introduction of a set of AFP transgenes that allow the fish to survive lower water temperatures, or (2) introduction of GH transgenes to produce a rapidly growing strain that does not require overwintering (Hew and others 1995). Although AFP transgenes have been successfully introduced into, expressed in, and inherited through germlines of Atlantic salmon, the cold-tolerant transgenic salmon do not produce AFP in sufficient quantities to achieve freeze resistance (Hew and Fletcher 1992; Hew and others 1995; Fletcher and others 2004). This may be due to the need for higher expression levels and/or the fact that the gene actually codes for an AFP precursor that must first be changed into its fully functional form (Maclean and Laight 2000). The current challenge, according to Fletcher and others (2004), is to create an AFP transgene construct that will be expressed at heightened levels in the epithelial tissues and the liver. 

Disease resistance. A major limitation facing the aquaculture industry is outbreak of disease, as farmed fish are generally cultured at high densities and under stress, putting them at elevated risk for bacterial infection (Hew and others 1995). One example can be found in the catfish industry, which suffers an average annual loss of over US$100 million because of disease (Dunham and others 2002b). Channel catfish (Ictalurus punctatus) are an economically important species in the United States, accounting for more than 60% of U.S. aquaculture production (269000 metric tons per year) (USDA 2001). However, these fish are susceptible to numerous harmful bacterial infections, the most detrimental being enteric septicemia, caused by Edwardsiella ictalurii (Dunham and others 2002b). Antibiotics can help provide disease resistance, but only a limited number have been approved for use in aquaculture (Sarmasik and others 2002). Although there are effective vaccines available for some diseases, many common catfish diseases, including enteric septicemia, do not have truly effective treatment methods (Dunham and others 2002b). Also, use of DNA vaccines is often labor-intensive and can cause high stress to the fish owing to excessive handling (Sarmasik and others 2002). A promising alternative involves use of transgenic technology to produce strains of fish with increased disease resistance. A number of antimicrobial peptides with the potential to improve disease resistance in aquaculture have been isolated from fish. The gene coding for these peptides has not been well characterized; however, a few possibilities are discussed below. 

Cecropins are a group of small, antibacterial peptides first identified in the silk moth Hyalophora cecropia (Dunham and others 2002b). Cecropins have antimicrobial activity against a wide spectrum of bacteria and have already been incorporated into transgenic plants such as potato and tobacco to provide increased disease resistance (Sarmasik and others 2002). Channel catfish with transgenically introduced cecropin genes demonstrated increased disease resistance and survival when exposed to E. ictalurii and Flavobacterium columnare, as compared to nontransgenic controls (Dunham and others 2002b). Transgenic individuals exposed to F. columnare in an earthen pond showed 100% survival, which was significantly greater than the nontransgenic controls (27.3% survival). When challenged with E. ictalurii in tanks, transgenic individuals also showed significantly greater survival (40.7%) as compared to nontransgenic controls (14.8%) (Dunham 2005). Transgenic Japanese medaka (Oryzias latipes) containing insect cecropin or pig cecropin-like peptide transgenes driven by a CMV promoter have also shown enhanced bactericidal activity against 2 known fish pathogens, Pseudomonas fluorescens and Vibrio anguillarum (Sarmasik and others 2002). Cecropin transgene constructs were electroporated into medaka eggs, and survivors containing the transgene were used as founder stocks to establish successive generations of transgenic medaka. When the F2 transgenics were exposed to P. fluorescens and V. anguillarum at 60% lethal dose, only 10% and 10% to 30% were killed, respectively, while 40% of the controls were killed by both pathogens (Sarmasik and others 2002). 

Lysozyme is a nonspecific antibacterial enzyme present in the blood, mucus, kidney, and lymphomyeloid tissues in fish (Hew and others 1995). Rainbow trout contain elevated levels of lysozyme (10- to 20-fold higher than in Atlantic salmon) and a rainbow trout lysozyme cDNA construct with an ocean pout AFP promoter has been created (Hew and others 1995). Interestingly, rainbow trout were recently reported to have 2 distinct types of lysozymes, with only type II having significant bactericidal activity (Mitra and others 2003). The gene for type II lysozyme was amplified and sequenced for future use in transgenic immune system enhancement of farmed fish. Lysozyme transgenes are also being tested in agricultural products such as rice and have been found to increase disease resistance (Huang and others 2002). Although the lysozyme gene may prove to heighten disease resistance in transgenic fish, research is still in the initial phases with no published results to date (Fletcher and others 2004). 

Human lactoferrin (hLF) is a nonspecific antimicrobial and immunomodulatory iron-binding protein that has been used widely in agriculture for production of disease-resistant transgenic crops, including potatoes and tobacco (Mao and others 2004). One use of hLF in fish is to increase resistance against the grass carp hemorrhage virus (Zhong and others 2002). This virus induces deadly hemorrhaging in grass carp and is a major setback to the successful farming of these fish in China. To induce resistance against the virus, a DNA construct containing the hLF gene linked to a common carp β-actin promoter was electroporated into the sperm of grass carp (Ctenopharyngodon idellus) (Zhong and others 2002). Gene transfer efficiency was reported to be near 50%, and after 5 mo about 36% of the surviving grass carp contained the transgene. After being challenged with the virus, transgenic fry showed a significant delay in the onset of symptoms of hemorrhage, indicating a possible use for hLF gene expression in grass carp. In a more recent study, electroporation of hLF cDNA into grass carp sperm resulted in as high as 55% production of transgenic fish (Mao and others 2004). These fish had increased immunity against infection with the bacterial pathogen Aeromonas hydrophila as compared to nontransgenic controls; transgenic grass carp had enhanced phagocytic activities and were able to clear A. hydrophila from their systems more quickly (Mao and others 2004). The authors hypothesized that hLF increases disease resistance by stimulating phagocytic activity in transgenic fish. 

A series of recent studies have focused on the use of shark DNA to boost immune responses in fish (El-Zaeem and Assem 2004; Assem and El-Zaeem 2005). Sharks contain high levels of immunoglobulin (IgM) proteins, which act as antibodies and help initiate immune responses to bacterial invasions. Although IgM can be found at high levels in shark (up to 50% of the serum proteins), it has been reported to be present at much lower levels in fish such as Atlantic salmon, halibut (Hippoglossus hippoglossus L.), haddock (Melanogrammus aeglefinus L.), and cod (Gadus morhua L.) (2%, 8%, 13%, and 20% of the serum proteins, respectively) (Marchalonis and others 1993; Magnadottir 1998). When shark (Squalus acanthias L.) DNA was injected into the skeletal muscles of Nile tilapia (O. niloticus) and redbelly tilapia (Tilapia zillii) fingerlings, fish showed significantly higher levels of total antibody activity, serum total protein, and globulin (El-Zaeem and Assem 2004; Assem and El-Zaeem 2005). In addition, injected tilapia had significant growth enhancement and changes in proximate composition, with decreases in moisture and increases in both protein and lipid content. Injected fish showed high genetic polymorphism, indicating random integration of the shark genes into tilapia muscle DNA. The highest injection dose resulted in deformities in the ovaries and testes of tilapia, suggesting a negative effect on spawning and reproductive abilities. Consequently, the authors pointed to a need for further studies into the effects of injected DNA on following generations. 

An additional biotechnological application in the aquaculture industry is the treatment of fish with poly I:C, a potent inducer of type I interferons (IFNs) (Jensen and others 2002). Type I IFNs are known to stimulate expression of myxovirus resistance (Mx) proteins, which are GTPases that inhibit the replication of single-stranded RNA viruses such as infectious salmon anemia virus (ISAV), one of the most economically harmful pathogens in the Atlantic salmon industry. When challenged with ISAV, Atlantic salmon treated with poly I:C experienced increased levels of Mx proteins and reduced mortality as compared to untreated controls (Jensen and others 2002). Since Mx proteins have been successfully cloned from Atlantic salmon, Japanese flounder, rainbow trout, and Atlantic halibut, they could potentially be introduced into transgenic fish. These fish could then be treated with poly I:C in order to induce type I IFNs and Mx protein expression, thereby promoting resistance against pathogens such as ISAV. 

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