By Dr. Dave Beatty
Written for Spring 2016 Fish Tales
Salmon Science: The Story of a Transgenic Salmon (Genetically Modified Organism)
Consumers in the US can expect to find transgenic Atlantic salmon for sale in the market within a few years. Here is an abridged story about how it all happened.
The Basics
A transgenic organism, or genetically modified organism (GMO), is produced when the biotechnology (recombinant DNA or rDNA technology) of genetic engineering is used to insert and stably integrate a gene or DNA sequence (a transgene) into the genome of a host organism (transgenic organism).
The transgene can occur in the host’s offspring. The biotechnology for producing a transgenic organism can rapidly alter its genome compared to the slow process of genetic selection achieved through crossbreeding or hybridizing in matings of closely related organisms.
In a transgenic, the transgene is often from an organism distantly related to the host or even totally unrelated taxonomically. Very often, transgenic organisms are produced in laboratories for research purposes. However, the production of transgenics is often for commercial purposes.
Chinook + Pout
In 1989, a group of scientists in Newfoundland, Canada, did the initial research to develop a two species rDNA construct (a transgene) by linking the growth hormone gene of Chinook salmon with the antifreeze protein gene from a species of ocean pout (an eelpout of the cold Northwest Atlantic Ocean).
When the transgene construct was microinjected into fertilized eggs of Atlantic salmon (AS), it integrated into the Atlantic salmon’s genome and transgenic Atlantic salmon (TAS) were produced. This founder TAS was backcrossed with wild-type Atlantic salmon and the progeny had the identical transgene construct demonstrating that the transgene is reproductively stable.
The ocean pout has antifreeze proteins in its body fluids to survive in subzero Celsius seawater. The ocean pout antifreeze gene acts as a promoter to stimulate Chinook growth hormone (CGH) production and release throughout the year in the TAS, even in very cold water. Many of the TAS had growth rates 2 to 6 times the growth rate of wild type AS controls.
In Chinook salmon, its natural promoter for CGH is active seasonally in response to specific environmental cues (e.g., day length and water temperature and growth rate responds accordingly). Likewise in normal AS, GH production is reduced in colder water as is the growth rate.
Three Concerns
AquaBounty Technology, a biotechnology company in Massachusetts involved in research and development directed at increasing productivity in aquaculture, recognized the opportunity of developing TAS for commercial aquaculture. Compared to conventional farm raised AS in Norway, North America, Great Britain and Chile, the TAS would have at least twice the growth rate, reach market size at a younger age and might have a higher efficiency of food converted into body mass.
AquaBounty continued to develop the technology to create its TAS as the AquAdvantage Salmon (AAS) and acquired the license for the technology. Salmon aquaculture companies in Norway and Chile spurned the production of TAS fearing consumer reaction, regulatory concerns and loss of market share. AquaBounty believes AAS produced in aquaculture has the potential to provide protein faster for an increasing world demand than can conventional aquaculture of AS.
Immediately, concerns were raised: 1) if AAS escaped and became established in the natural environment, would they interbreed with wild, native Atlantic salmon or otherwise adversely affect their ecosystems; 2) would AAS have significantly altered phenotypes (e.g., body shape and malformations, swimming ability and behavior in the confines of the aquaculture system); and 3) would the transgene construct have an effect during the AAS’s growth and development affecting human health if AAS are consumed?
AquaBounty, as it sought approval during the mid-1990s from the United States Food and Drug Administration’s Center for Veterinary Medicine to market AAS, submitted documentation addressing these concerns. The TAS broodstock and its transgenic progeny, the AAS, would have biological, physical and environmental constraints to prevent the escape of AAS into the environment and if escape occurred, to eliminate interbreeding with wild type AS and thereby prevent transmission of the transgene into wild populations.
1) Biological Containment
In the production of AAS, fertilized eggs are treated with high pressure within an hour after fertilization to produce triploid eggs (AquaBounty claims no more than 1.1% remain diploid). Wild salmon are diploids (two complete sets of chromosomes or 2N, one set from each parent), whereas triploids have three complete sets of chromosomes or 3N (two from the female parent and one from the male) resulting from the high pressure treatment of eggs.
Triploid females are sterile and do not produce eggs. However, some triploid males do produce sperm and thus may be fertile. Also, any diploid eggs (triploidy is not 100%) could produce diploid AAS of both sexes. If any of the triploid AAS males capable of producing sperm (not all are sterile) or of the diploid AAS escaped into the environment, they could mate with wild type AS and thereby introduce the transgene (“the alleged Trojan gene effect”) into a wild population of AS. Consequently, an additional method for reproductive isolation of the transgene was required to prevent the possibility of AAS matings with wild salmon if AAS escape and survive.
All TAS used to produce sperm for fertilizing Atlantic salmon eggs are neomales (reproductive males that are genetically female). To produce neomales, female TAS homozygous for the Chinook salmon growth hormone/sea pout GH promoter transgene construct (it is present on both chromosomes of a homologous pair) are treated with 17-methyltestosterone to create genetic females with testes to produce sperm and all sperm carry the transgene construct because of homozygosity for the transgene. Female salmon are XX (sex chromosome pair for sex determination) and the neomales (by sex reversal of females) are also XX for sex chromosomes whereas normal males are XY for the pair.
All progeny resulting from high pressure treatment of wild type Atlantic salmon eggs fertilized with sperm from transgenic neomales (sex reversed TAS females) produce nearly all triploid and a relatively small number of diploid female progeny, each having one copy of the transgene construct provided by sperm from the transgenic neomale. No males will be produced because there are no XY (the pair of sex chromosome for becoming male) males.
Consequently, the sex reversal of a female TAS homozygous for the transgene construct to produce neomales producing sperm containing the transgene and the pressure treatment of fertilized eggs to produce nearly all triploids results in sterile, triploid female AAS that will not produce eggs, or if eggs are produced they lack the genetic framework for fertilization.
This is the final biological containment. However, a small percentage (no more than 1.1% according to AquaBounty) of AAS can be diploids because the pressure treatment of fertilized eggs is not 100% for triploidy. If the diploid AAS females (no males exist) escaped, they could mate with wild salmon. Therefore, physical containment of AAS is necessary.
2) Physical Containment
Closed containment with no possibility of escape or accidental release of eggs or AAS is essential even if the capability of these transgenics to survive in natural ecosystems and to interbreed with wild salmon is very small. Currently, all fertilized AAS eggs are produced from broodstock held in tightly controlled, closed containment facilities in Prince Edward Island, Canada. When the AAS eggs develop to the eyed stage and can survive transport, they are air shipped to a land-based rearing facility in a mountainous region of Panama. In November 2013, Canada approved the production of AAS eggs for commercial use.
3) Environmental Containment
The Panama facility is close to a river draining to the Pacific Ocean and thus there is the potential for introducing AAS into the Pacific Ocean. Although a developing embryo, juvenile or adult might escape and survive in the river, a juvenile or adult would likely not survive when it reached the warmer river water as it moved downstream. Likewise, any AAS reaching the ocean would meet lethal temperatures.
AquaBounty believes it has built in a redundancy of protections to avoid AAS’s introduction into wild AS ecosystems and thereby eliminate mating with wild fish.
Transgenic vs. Wild Studies
Biological and ecological risk assessments must be done in closed containment facilities and should be done under as wide a range of conditions as feasible.
Intense studies to compare transgenic salmon to wild salmon have been done at the Fisheries and Oceans Canada Laboratory in West Vancouver. The studies used growth enhanced transgenic coho salmon (TCS) whose genome contains the transgene construct of a promoter driving the expression of a sockeye salmon growth hormone gene. The TCS are then compared to hatchery coho (HC) at different life stages under a variety of experimental conditions.
As an example, in a study on the reproductive performance of TCS in a simulated natural environment, the TCS show courtship behavior, can spawn together or with HC and produce viable TCS progeny.
Because wild type TCS are not available, it is not possible to fully determine the TCS’s reproductive fitness. However, these studies demonstrate the capability of transgenic salmon to mate with wild salmon. Also, the research provides evidence for the transmission of a transgene to wild populations if mature transgenic salmon are in contact with mature wild salmon of the same species.
Consumption Concerns
AquaBounty received mounting criticism from numerous consumer advocacy groups once it was known of the intent to market the AAS. Allergenicity and possible adverse changes in the nature of the edible flesh, including increased levels of specific hormones of potential human consequence, have been major concerns.
Specific to AAS is the transgene construct derived from integrating Chinook salmon GH and the sea pout promoter and triploidy. Certain individuals are known to be allergic to wild type AS and thus are likely to have an allergic reaction to AAS. However, would the allergenicity be more intense or even expand to more individuals?
Using high pressure treatment of fertilized eggs, hatcheries produce and release triploid rainbow trout (not transgenic) for recreational fishing and possible consumption. There is no evidence indicating triploid trout are more allergenic than wild type diploid trout.
FDA Approval
When FDA’s Center for Veterinary Medicine began the process to determine whether AAS is safe for human consumption, the guidelines for such a determination were not fully developed. Why was FDA involved? The AAS transgene construct is considered a drug for use in an animal and thus AquaBounty had submitted a New Animal Drug Application (NADA).
In 2010, FDA determined that AquaBounty had provided sufficient information to make its decision. The critics’ opposition mounted over several shortcomings in the fundamental information AquaBounty provided, especially for the allergenicity and the amount of a specific hormone in the flesh.
Furthermore, in the FDA’s process for determining the safety of AAS, FDA had not consulted NOAA’s National Marine Fisheries Service and the U.S. Fish & Wildlife Service regarding the potential biological and environmental effects of the AAS on endangered Atlantic salmon. In 2001, NMFS and USFWS issued a Biological Opinion to the U.S. Environmental Protection Agency to ban all transgenic salmon on ocean-based salmon farms, a ban that FDA seemed to ignore in 2010 while AquaBounty continued to consider this option for rearing AAS. Consequently, a full Environmental Impact Statement was required. In 2010, several members of Congress requested the FDA to reject the approval of AquaBounty’s application.
For another five years, the FDA approval process continued. In December 2012, FDA published a draft Environmental Assessment for AAS and also published a “Finding of No Significant Impact.” In May 2013, the public comment officially ended and FDA could proceed to complete its assessment.
On Nov. 19, 2015, FDA gave approval to AquaBounty’s NADA for the production and marketing of AAS with conditions and restrictions including 1) all AAS eggs shipped for rearing must be triploid eyed eggs produced at the Prince Edward Island facility and are to be used only by AquaBounty at its Panama rearing facility and 2) any purchase or use of AAS eggs by a third party or any rearing of AAS at other than the Panama facility would require a new application.
For a comprehensive review, see Alain Goubau’s “The AquAdvantage Salmon Controversy - A Tale of Aquaculture, Genetically Engineered Fish and Regulatory Uncertainty” (May 2011) at https://dash.harvard.edu/handle/1/8789564.
For FDA’s AquAdvantage Salmon Approval Letter and Appendix for the extensive restrictions and conditions, see http://tinyurl.com/j6at2yz.