Modern biotechnological advancement has come a long way since the monumental discovery of DNA, allowing humans to alter and rewrite the genetic encoding of life. Considered the most impressive and dynamic biotechnological application is the transfer of constructed gene assemblies from laboratory to field organisms. In essence, this genetic engineering enables individual genes controlling certain characteristics to be separated from their genetic origin and transferred directly into the genome of individuals of the same species or unrelated species. The resulting ‘artificial’ animal, plant, bacterium, or virus are termed genetically modified organisms (GMOs) (Prakash et al. 2011). This technology has a wide range of potential environmental applications: sustainable agriculture, forestry, aquaculture, biocontrol of diseases, biopesticides, bioremediation, improvisation of nutritional quality of food, and many more in both developed and developing countries (Snow et al. 2005). Genetically modified crops, in fact, have been the most rapidly introduced and accepted technological practice in the history of agriculture and food science (Singh, 2006); however, when introduced into the natural environment, GMOs have the ability to cause unintended environmental consequences. If genetically engineered organisms are able to reproduce and establish persistent populations, subtle and long-term effects on adjacent biological communities and the ecosystem as a whole may be observed, especially if the recombinant form has a more pronounced ecological role compared to its wild type (Prakash et al. 2011). Genetic engineering could have a profound impact on the natural environment; therefore, a cautious approach is necessary to assess potential risks to the introduction of recombinant organisms in the natural environment. This paper will address three critical issues at the center of this subject: an assessment of the potential hazards of environmental release in terms of the modes of gene transfer between GEOs and wild types, a review of modern GEOs and their observed impacts, and a review of the methodologies established to ensure biosafety after release.
Altering the genetic code of a life form can have pronounced effects on all aspects surrounding it, especially once removed from a controlled laboratory setting and placed into the will of nature. Possible risks of the release of genetically modified organisms include: (1) gene pool contamination through the horizontal and vertical gene transfer of recombinant genes, which could lead to hybridization, the conference of novel traits, and the creation of biological resistance and impossibility of containment, (2) eradication of natural species and irreversible loss of genetic and biological diversity, and (3) ecological and zoonotic health threats (Keese 2008, Prakash et al. 2011).
Transfer of genetic information is achieved by one of two mechanisms: vertical or horizontal gene transfer. Vertical gene transfer (VGT) is the direct transfer of genetic material from parent to offspring by way of sexual compatibility or asexual reproduction (Steinbrecher & Latham et al. 2003). Horizontal gene transfer (HGT) is the transfer of genetic material from one organism to another of the same or different species without reproduction (Lorenzo-Diaz et al. 2017). HGT occurs by the passage of genetic material across cellular boundaries, followed by incorporation in the genome of the recipient. This can occur naturally or by human intervention (e.g. genetic engineering, in vitro fertilization, protoplast fusion, self-cloning, etc.) (Keese 2008). HGT functions to spread multiple genetic traits and actively contributes to the adaptation of bacteria to new niches which can lead to the optimization of antibiotic, insecticide, herbicide, and other resistances (Lanza et al. 2015, Smillie et al. 2010). Further, growing evidence suggests that HGT influences not only prokaryotic life, but also impacts the evolution of complex, eukaryotic organisms, suggesting that genetic information can be transferred between them (Schaack et al. 2010). The transfer of antibiotic resistance genes to a pathogen can have dramatic effects on human and animal health and medicine. Controversially, the genetic transfer to humans has the potential to prompt oncogenesis (Ho et al. 2009) and the transfer of viral transgenes to unrelated viruses has the potential to prompt novel, emerging diseases (Falk & Bruening 1994).
More recently, major concerns have been raised regarding the transfer of genetic material between GMOs and their natural types through these mechanisms of gene transfer. Interspecific hybridization, especially, is a plausible route for transgenes of altered organisms to invade wild type populations, potentially creating an unsustainable hybrid species by way of vertical gene transfer (Oke et al. 2013).
Oke, Westley, Moreau, and Fleming, through experimental crosses, made the first known demonstration showing the environmental impacts of hybridization between a GE animal and closely related species in 2013. They proved that the transmission of a growth hormone transgene via vertical gene transfer (hybridization) between a GM Atlantic salmon (Salmo salar)—a candidate for commercial aquaculture, and a closely related species of wild brown trout (Salmo trutta) was a viable possibility. In fact, transgenic hybrids grew more rapidly than both originating species in a hatchery setting, and displayed competitive dominance over them in a simulated, natural stream setting. This hybrid species suppressed the growth of both the GM salmon and wild-type salmon by 82 and 54 percent, respectively; thus, the hybridization between a GM Atlantic salmon and a wild brown trout could lead to outgrowth effects in a natural setting, eradicating related species, and pose ecological threats to biodiversity and the trophic cascade. Interspecific hybridization must be considered when making assessments on environmental consequences of transgenic species (Oke et al. 2013) and preventative mechanisms must be established and maintained.
Beginning in 1989, research on an AquAdvantage® Salmon species commenced to develop the first genetically modified food animal for human consumption. AquAdvantage® Salmon were developed by transgenically inserting a growth hormone extracted from the genome of a related Chinook salmon species into a fertilized Atlantic salmon egg (α-form of the opAFP-GHc2 rDNA at the α-locus in the EO-1α) (Dunham 2017). Engineered salmon displayed a rapid-growth phenotype, allowing them to reach market size (~100 g) in half the time of conventional, non-engineered, farm-raised Atlantic salmon, making them an optimum alternative for both commercial aquaculture enterprise and nutritional quantity (Clifford 2014, U.S. Food and Drug Administration 2018). To prevent the vertical gene transfer of the transgenically inserted growth hormone, AquAdvantage® Salmon are specifically designed to be an all female, triploid, sterile species maintained in a freshwater, land-based containment facility (Clifford 2014). This would prevent interaction and sexual compatibility with other species, eliminating hybridization. In November of 2015, the Environmental Assessment (EA) of this New Animal Drug Application (NADA) was reviewed and approved under the Federal Food, Drug, and Cosmetic Act (FD&C Act) and environmental impact considerations regulations of the Food and Drug Administration (FDA), by the FDA and Center for Veterinary Medicine (CVM). The FDA issued a Finding of No Significant Impact (FONSI), concluding that, under the specified conditions of this NADA, the likelihood and consequence of AquAdvantage® Salmon escaping and becoming established in a natural setting is extremely low and, individually or cumulatively, this GM species would not have a significant ecological or human health impact in the United States (Center for Veterinary Medicine et al. 2016). The multiple active, physical and biological containment measures ensure the absence of VGT and biosafety of this genetically engineered organism.
Aedes mosquitoes vector positive-sense RNA flaviviruses, including dengue virus, West Nile virus, and yellow fever virus; all highly pathogenic to humans (Skalsky et al. 2010, Garziera et al. 2017) so the inspection of GM techniques to offer solutions to these viral epidemics is crucial, but the potential impacts on human and animal health without strict management include toxic or allergenic effects in humans and other non-target species, unexpected transfer of the transgene, and an increase in disease vectoring or antimicrobial resistance. A study was performed by Oxitec, Ltd. proposing a GE male Aedes aegypti mosquito of the line OX513A encoding a conditional lethality trait and a fluorescent red marker protein for identification with the goal of evaluating the breeding potential of OX513A males with wild-type Aedes aegypti and the survival potential of resulting offspring. The potentials for HGT of transgene via prokaryotes to other prokaryotes and eukaryotes were also evaluated, as well as the suppression of overall Aedes aegypti populations in the test area (Center for Veterinary Medicine et al. 2016). The lethality trait is a function of the overexpression of the tetracycline-repressible transactivator (tTAV) protein. In the absence of tetracycline in the GM mosquito habitat, the tTAV protein causes mortality in those with at least one copy of the recombinant DNA transgene, which includes any hybridized progeny. Oxitec, Ltd. provided information and supporting data for its proposed field trials in Florida using only GE male Aedes mosquitoes to the FDA and CVM. Under the strict regulations of this experiment and multiple physical, biochemical, and genetic barriers, the FDA found that the probability of adverse effects on human and non-targeted species health is extremely low and the risk is negligible since male mosquitoes are not capable of transmitting disease via bite. The FDA also determined that the likelihood of the OX513 rDNA transgene being transferred to other insects, animals, humans, and microorganisms via VGT or HGT is extremely low (Center for Veterinary Medicine et al. 2016). The stable integration of the transgene into the mosquito genome and the impossibility of the remobilization of the transgene, even when treated with appropriate transposases, ensure the risk is negligible. It was also determined that the probability of the production of antimicrobial resistance in adult OX513 adults is extremely low due to the intestinal (gut) bacteria being lost during metamorphosis. Further, it was concluded highly unlikely that GM males would interbreed with other mosquito wild-type species resulting in hybridization as mosquito mating is species-specific. The FDA and CVM found all risks behind the introduction of this proposed GM species to be negligible.
The FDA’s analysis of the aforementioned GM species in both EAs is based on the characterization of potential hazards, exposure pathways and receptors; the estimation of risks and the uncertainty of these estimates. In both cases, strict regulations, including physical, biochemical, and genetic obstacles, were implemented to successfully prevent the genetic transfer between genetically engineered organisms and wild-types in a natural environment.
It has been proven that, whether in an aquatic or terrestrial environment, GMOs can have a profound effect on the biodiversity, primary production, trophic level relationships, and ecological function and balance. In view of this, it is necessary for biosafety regulations to be established, and risk analyses to be premeditated and performed before any introduction of genetically modified organisms can be successful or accepted. Risk is a function of hazard and exposure. If the exposure and transmission of engineered genomes is negligible, thus will be the risk.