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4 Bioconfinement of Animals: Fish, Shellfish, and Insects This chapter focuses on bioconfinement of two broad categories of genetically engineered organisms (GEOs): aquatic animals and insects. The aquatic animals considered are finfish (trout, catfish, tilapia) and shellfish, including mollusks (oysters, clams) and crustaceans (shrimp, crayfish). The Committee on the Biological Confinement of Genetically Engineered Organ- isms chose to focus on fish, shellfish, and insects because they are highly prone to establishing feral populations if they are intentionally introduced into the environment or if they escape from aquacultural or agricultural systems (NRC, 2002b). Captive lineages of those animals might serve as founders for genetically engineered lines, but they have undergone so little domestication that they often can reproduce and survive in suitable natural environments. Their reproductive and ecological traits are closely related to those of their wild relatives, thus raising the possibility of gene flow to or competition with wild relatives. Furthermore, many of the species of fish, shellfish, and insects targeted for genetic engineering have wild relatives in the environments they are likely to enter. The chapter does not explicitly address bioconfinement of terrestrial livestock species because, as a group, they are less prone to becoming feral and causing ecological problems. There have been some important excep- tions, however, such as feral goats in many countries and pigs, range chickens, and turkeys in several U.S. states (NRC, 2002b). When contem- plating genetic engineering of livestock species that can become feral, it will be important to consider options for bioconfinement as part of the mix of feasible confinement methods. Some of the general approaches discussed in 130

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ANIMALS: FISH, SHELLFISH, AND INSECTS 131 this chapter, such as the regulation of gene expression to prevent successful reproduction of escaped adults, also could be applied to livestock, although they would require tailoring to the biology of the species at issue. The committee's major findings and recommendations therefore apply generally to terrestrial livestock species that are prone to becoming feral. Furthermore, this chapter does not directly address bioconfinement of laboratory research animals, such as inbred strains of mice or rats. Labora- tory animal strains typically are held in rearing or research facilities with multiple physical containment features and high security against theft. If research with transgenic lines of laboratory animals were to rely more heavily on bioconfinement than on physical confinement, the committee's findings and recommendations also would apply generally to those species (cat, mink) that might escape the laboratory and become feral in an accessible ecosystem. Biotechnologists are developing transgenic fish and shellfish for a diver- sity of purposes (Table 2-2; Kapuscinski, 2003, and references therein). The proposed application of many transgenic lines is in aquaculture to produce human food, and it focuses on increasing growth rates and food conversion efficiency or improving disease resistance. Scientists also are developing transgenic lines for use as biofactories to produce pharmaceuticals, indus- trial chemicals, or dietary supplements; in bioremediation to remove con- taminants from water; as water quality sentinels to detect contaminants that damage the genes of living organisms; and for biological control of nuisance aquatic species. Some degree of mechanical and physical confine- ment is possible for some of the proposed transgenic fish and shellfish (Scientists' Working Group on Biosafety, 1998). In other cases transgenic lines will be introduced into natural waters, either deliberately as in biological control applications, or unintentionally by escape from floating net cages, outdoor ponds in flood-prone zones, and flow-through raceways. One also can envision proposals to deliberately release hatchery-propagated fish or shellfish, such as cold-tolerant or endemic-pathogen-resistant lines, to estab- lish a new sport or commercial fishery, or to augment an existing fishery. There are several reasons for developing genetically engineered insects. Agricultural applications include transgenic-based sterile males (replacing radiation-induced sterile males) for mass releases in biological control of pest insects, visual transgenic marking with markers such as green fluores- cent protein that can be used to evaluate effectiveness of sterile insect releases, and genetic engineering of beneficial insects (predators and parasi- toids of pest insects) for resistance to insecticides to allow simultaneous use of both methods of controlling pest insects (Braig and Yan, 2002; NRC, 2002b; Wimmer, 2003). Genetic engineering also has been proposed for introducing disease resistance and other desirable traits into domesticated insects, such as honeybees and silkworms, and to turn insects into biological

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132 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS factories for mass production of valuable proteins such as collagen, which could be produced by silkworms (Tamita et al., 2003). Finally, genetic engineering research is under way to disrupt transmission of diseases by mosquitoes and other vectors (Braig and Yan, 2002; NRC, 2002b; Spielman et al., 2002). In this last application, bioconfinement is not an option because achievement of disease suppression requires that the released transgenic insects mate widely with wild-types to spread their transgenes throughout the population. The discussion in this chapter assumes that the transgenic animals are dioecious--male and female reproductive organs are in separate individuals and each individual is of one sex throughout its lifetime. However, non- dioecious modes of reproduction, such as hermaphroditism and partheno- genesis, occur in some species of fish, mollusks, and crustaceans, some of them aquaculturally important (reviewed in Appendix B of ABRAC, 1995). Bioconfinement methods discussed in this chapter that target sexual repro- duction could fail to achieve the desired amount of confinement or, in some cases, could simply be infeasible in hermaphroditic and parthenogenetic species. Hermaphroditic individuals have male and female organs; parthenogens have some form of clonal inheritance of genomes (Moore, 1984). Hermaphro- dites occur in some species of sea bream (Buxton and Garrett, 1990), a family of finfish with several species produced in aquaculture, and at least one species that is already the subject of gene transfer for growth enhance- ment (Zhang et al., 1998) and reported to exhibit hermaphroditism (Huang et al., 1974). Parthenogenesis occurs in strains of aquacultural crustaceans such as Artemia (brine shrimp) (Triantaphyllidis et al., 1993) and Daphnia spp. (Hebert et al., 1993). Self-fertilizing hermaphrodites and true parthenogens, which do not require the physical stimulus of sperm to induce embryogenesis, pose the greatest challenge for confinement because the escape of just one fertile individual could result in the establishment of an entire population. BIOCONFINEMENT OF FISH AND SHELLFISH Bioconfinement methods currently in practice for fish and shellfish either reduce the spread of transgenes and transgenic traits through disrup- tion of sexual reproduction or rely on ecological characteristics of the production site that are lethal to some life stage of an escaping organism. Disruption of Sexual Reproduction Methods for disruption of sexual reproduction include induction of triploidy or interploid triploidy--causing embryos that normally bear two

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ANIMALS: FISH, SHELLFISH, AND INSECTS 133 sets of chromosomes to carry a third set; induction of monosex lines; and crossing two closely related species to produce viable but infertile hybrids (sterile, interspecific hybrids). The methods sometimes are combined, par- ticularly triploidy monosex production. Sterilization through Induction of Triploidy Triploidy induction involves application of hydrostatic pressure or temperature or chemical shock at the appropriate number of minutes after egg fertilization to disrupt the egg's normal extrusion of a polar body that contains a haploid set of chromosomes. The resulting retention of the polar body leads to an embryo that bears a pair of haploid chromosome sets from the female (instead of the normal single set) and a third set from the male (Figure 4-1). The presence of the odd set of chromosomes presumably causes mechanical problems involving the pairing of homologous chromo- somes during each cell division (Benfey, 1999), and this disrupts the normal development of gametes to some extent, as explained below. Triploidization is much better developed for finfish and mollusks than it is for crustaceans produced in aquaculture. Protocols for large-scale induction of triploidy have been worked out for a number of commercially important fish and mollusks, including various trout and salmon species, channel catfish, African catfish, various tilapia species, various carp species, oysters, and clams (reviewed in Beaumont and Fairbrother, 1991; Benfey, 1999; Li et al., 2003; Tave, 1993; Thorgaard, 1995). However, protocols need to be developed and optimized for each species. Induction of triploidy in crustaceans might be possible only in shrimp species that spawn free eggs (genera Litopenaeus and Penaeus) and not in those species, such as fresh- water prawns (Macrobrachium rosenbergii), whose females incubate their fertilized eggs (Beaumont and Fairbrother, 1991; Dumas and Campos Ramos, 1999). Researchers are in the early stages of developing reliable protocols for triploid induction in marine shrimp, but recent efforts are part of the increased interest in the genetic improvement of shrimp--from tradi- tional breeding to gene transfer (e.g., Dumas and Campos Ramos, 1999; Fast and Menasveta, 2000; Li et al., 2003). Strengths Triploidy induction has become widely accepted as the most effective method today for producing sterile fish for aquaculture (Benfey, 1999; Tave, 1993). It is the best-developed method of disrupting sexual reproduc- tion, and it has the most complete scientific documentation of strengths and weaknesses. Triploidy has been used on commercial rainbow trout and Atlantic salmon farms (Donaldson and Devlin, 1996). Triploid Pacific

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134 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS Spawning Polar Body Extrusion Fertilization Zygote Chromosome Duplication Cell Division Chromosome Duplication Cell Division DIPLOID (2n) TETRAPLOID (4n) TRIPLOID (3n) FIGURE 4-1 Normal steps in gamete fertilization and early cell division that lead to the development of a normal diploid (2n) fish or shellfish embryo. Induction of triploidy (3n) or tetraploidy (4n) occurs by temperature shock, chemical shock, or pressure at an appropriate time after fertilization: denotes the point at which the shock is applied; denotes one haploid chromosome set derived from the female parent; and + denotes one haploid chromosome set derived from the male. SOURCE: Adapted from Donaldson, unpublished data.

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ANIMALS: FISH, SHELLFISH, AND INSECTS 135 oysters make up 30% of all Pacific oysters farmed on the West Coast of North America (Nell, 2002), not so much for bioconfinement as to prevent yield losses associated with sexual maturation in production animals. Pro- cedures for inducing triploidy are easy to learn and require relatively inexpensive, simple equipment. It is feasible to screen individuals nonlethally and to collect blood, hemolymph (the shellfish equivalent of blood), or another small tissue sample, for the presence or absence of the triploid condition (Harrell and Van Heukelem, 1998; Nell, 2002; Wattendorf, 1986). Individual screening has long been required for large-scale stocking of triploid grass carp in Florida (Griffin, 1991; Wattendorf and Phillippy, 1996). Farmers interested in stocking this alien species into their irrigation canals to help control aquatic nuisance weeds are required to have each fish tested and certified as triploid before release. Weaknesses The incomplete success in producing triploids is a major problem, particularly for treating large batches of newly fertilized eggs. Several limi- tations to screening and detection affect success with culling individuals that fail to become triploid. The degree of functional sterility in triploids varies, depending on the species and sex of the fish. A small percentage of mosaic individuals (bearing a mix of diploid and triploid cells) also can compromise sterility if their gonads are diploid and thus develop into normal, fertile gametes. Sterile individuals that still enter into courtship behavior could disrupt successful reproduction of wild relatives, and recur- ring large escapes of sterile individuals could heighten competition with or predation on wild species. Commercial aquaculturists could resist adopting sterile lines of fish and shellfish. These weaknesses and possible mitigation are explained more fully below. Variable atriploidy: The percentage of triploids produced from a treated batch of eggs varies greatly by species and strain, method, pretreatment water temperature (when induction is by heat shock), and egg quality (see review in Galbreath and Samples, 2000). Reported success rates in finfish range from 10% to 100% (Galbreath and Samples, 2000; Johnstone et al., 1989; Maclean and Laight, 2000). Although little has been published about large-scale treatments, Johnstone and colleagues (1989) reported 100% triploid fish with a 90% survival rate relative to controls in a large-scale trial involving pressure shock on 50,000 eggs per hour. Commercial aqua- culture companies that produce and market triploid fish are likely to have closely held data on success rates of large-scale pressure shock and tempera- ture shock treatments for triploid induction. Effectiveness in shellfish ranges from 85% to 95% in oysters (personal communication, S. Allen, School of Marine Science, Virginia Institute of Marine Science, Gloucester Point,

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136 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS 2003) and from 63% to 100% on application of the "optimum" protocol in one shrimp species (Li et al., 2003). Screening to mitigate failed triploidization: Less than total triploid induction can be mitigated by screening treated individuals and then remov- ing the nontriploids before they are transferred from hatcheries to much less secure grow-out facilities, such as outdoor ponds or open-water cages (Kapuscinski, 2001; Kapuscinski and Brister, 2001). Mass screening is feasible through particle analysis or flow cytometry; particle analysis allows almost instantaneous results (Harrell and Van Heukelem, 1998; Nell, 2002; Wattendorf, 1986). Both methods permit non-lethal screening of larger juvenile life stages because they require only minute quantities of blood (as little as 1 L or one drop) or disaggregated tissue (Harrell and Van Heukelem, 1998). Detection limits and operator error are facts of life for either method. The critical management issue regarding the amount and verifiability of the bioconfinement provided by induction of triploidy is whether to screen every individual destined for grow-out or only a sample of each production lot. Such a decision should consider the risk, severity of consequence (Table 2-1 and Figure 2-1, Chapter 2), and the extent to which adequate additional confinement measures are in place. The discussion of transgenic salmon presented in Box 4-1 illustrates the point. Use of Tetraploids to Maximize Triploid Percentage The failure rate in producing triploid individuals can be reduced or avoided altogether by making triploid individuals via crosses between a tetraploid adult (usually the female) and a diploid adult (Guo et al., 1996; Tave, 1993; Xiang et al., 1993). Newly fertilized embryos are induced to become tetraploid (bearing four sets of chromosomes instead of the normal two sets) in the first generation (Figure 4-1). Then the diploid eggs pro- duced by a tetraploid female are crossed with the normal haploid sperm of a male to generate all-triploid offspring in the next generation. The off- spring are called interploid triploids, or "genetic" triploids, to distinguish them from induced triploids. The generally poor survival and performance of tetraploid fish (Donaldson and Devlin, 1996), however, prevents large numbers of individuals from reaching sexual maturity. This has discour- aged large-scale production of interploid triploids in finfish and could be an obstacle for bioconfinement of genetically engineered species. Much better performance of tetraploids has been reported in oysters produced by cross- ing eggs from triploid females with sperm from diploids (Allen and Guo, 1998). Most important for bioconfinement, the yield of interploid triploid oysters can be very high; one researcher has reported that 99.3% of more that 2,100 offspring were triploid (S. Allen, unpublished data), and the approach is in use by some commercial oyster farms (Nell, 2002). The

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ANIMALS: FISH, SHELLFISH, AND INSECTS 137 BOX 4-1 Proposed Bioconfinement of Transgenic Atlantic Salmon Aqua Bounty Farms, a biotechnology company, has applied to the U.S. Food and Drug Administration (FDA) for commercial approval of transgenic, growth- enhanced Atlantic salmon (Office of Science and Technology Policy and Council on Environmental Quality, 2001). The company intends to sell transgenic embryos or newly hatched fry to industrial salmon farms. The salmon farms would raise the juvenile fish in confined hatchery systems, usually consisting of land-based tanks and ponds, and then transfer the older smolts (a life stage that can thrive in sea- water) to less confined, floating cages in coastal marine waters. This has raised concerns about potential ecological harm, particularly to already severely depleted populations of wild Atlantic salmon. Introduction of a new threat to wild Atlantic salmon would occur in the face of costly and complicated efforts under way to recover declining Atlantic salmon populations (e.g., NRC, 2002d, 2004). Within the native range of Atlantic salmon, the primary ecological concern is whether the movement of transgenes into wild populations has a higher, equal, or lower potential to depress fitness (Kapuscinski and Brister, 2001; NRC, 2002b; Pew Initiative on Food and Biotechnology, 2003). Computer simulations have suggested scenarios involving earlier age at sexual maturity or larger size of repro- ducing adults--traits often associated with faster growth rates in fish--combined with moderately lower, equal, or higher viability in transgenic salmon than in wild fish, that could pose a heightened threat to the fitness of wild populations (Muir and Howard, 2001; NRC, 2002b; Pew Initiative on Food and Biotechnology, 2003). It is unclear whether the company has collected the data needed to assess whether its transgenic salmon fit any of these scenarios, partly because such data have not been reported in scientific journals and partly because of the lack of transparency in the FDA drug approval process (Kapuscinski, 2001; NRC, 2002b; Pew Initiative on Food and Biotechnology, 2003). In salmon farming regions outside the natural range of Atlantic salmon (e.g., Chile, New Zealand), the main question would be whether the net fitness of trans- genic salmon is higher or lower than in currently farmed strains and thus whether the transgenic fish would be more or less of a threat to invade native regions (NRC, 2002b; Pew Initiative on Food and Biotechnology, 2003). Heightened inva- siveness could pose a risk to native fish and other aquatic species through preda- tion or competition (Scientists' Working Group on Biosafety, 1998). The basis for concern is the increasing documentation of thousands to hun- dreds of thousands of farmed salmon that escape from cages that have been damaged by storms, predators, or wear and tear (e.g., Carr et al., 1997; Gross, 1998, 2001; Thomson, 1999). Most escapees are smolts, postsmolts, and adults; all of which can move from one habitat to another and interact directly or indirectly with wild salmon (NRC, 2002d, 2004). As they mature, escapees have been found to migrate into rivers (Hansen and Jonsson, 1991; Whoriskey and Carr, 2001; Youngson et al., 1997) and to spawn in those rivers (e.g. Clifford et al., 1998; Lura and Seagrov, 1991; Webb et al., 1991). Breeding between farmed salmon escap- ees and wild salmon can depress the reproductive success and competitive ability of wild populations through various mechanisms during the breeding season and in the next generation (NRC, 2002d, 2004). continued

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138 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS BOX 4-1 Continued A less-examined exposure route that could be significant (Stokesbury and LaCroix, 1997) is the escape of juvenile salmon from freshwater hatcheries operated by salmon-farming companies (NRC, 2002d). Competitive interactions between farmed and wild salmon juveniles for food and space in rivers can lead to displace- ment of wild fish and to depressed productivity of the wild population (Fleming et al., 2000; McGinnity et al., 1997). To reduce the likelihood of damage, Aqua Bounty Farms has suggested that it will sell nothing other than batches of embryos or newly hatched fry that are all- female and subjected to mass-scale induction of triploidy. That combination takes advantage of the fact that triploid salmon females cannot produce viable eggs even though triploid males can still produce viable sperm (reviewed above in this chapter). Resources for achieving strict confinement can focus on holding the transgenic broodstock needed to propagate the all-female progeny in one or a few facilities. The proposal also would protect the company's patent on the marketed line of transgenic fish by preventing salmon farmers from propagating the line because they would be required to purchase production fish for each grow-out cycle. The Aqua Bounty Farms proposal has two important weaknesses. First, it depends heavily on screening to identify and cull failures of triploid induction. The critical management issue is whether to screen every individual prior to transfer to grow-out facilities or only a sub-sample of each production lot, as discussed above in this chapter. Such a decision should consider the level of risk and severity of consequences (Table 2-1 and Figure 2-1, Chapter 2) and adequacy of the integrated confinement system (Chapter 6). The net fitness method (Muir and Howard 2001, 2002) provides a means to estimate--in a secure setting--the probability of spread of the transgenes if fertile transgenic salmon were to escape, although it cannot predict the severity of the harmful consequence from such transgene spread. This estimate would help decision makers determine whether to screen all or only a sub-sample of each production lot. If they choose sub-sampling, this estimate would help determine the appropriate sample size as a function of the predicted severity of harm, the probability of harm given an escape of fertile salmon has occurred, and the probability of escape of fertile fish. Individual screening followed by culling of diploids would be the more prudent choice for farming all-female, triploid transgenic Atlantic salmon in open-water cages in areas--such as the Maine coast--where wild populations are already depleted severely. Eight populations in Maine are listed as endangered under the terms of the Endangered Species Act (NRC, 2002d). Fewer than 100 sexually mature adults returned to these eight rivers in 20002002 (NRC, 2002d), and fish traps placed on three of the rivers intercepted up to 65 farmed salmon escapees each year (19932001). That number represents a range of 0100% of returning adults (NRC, 2002d). Those data suggest that even a small number of escaped fertile transgenic fish could constitute a major cohort of interbreeding adult fish in Maine's rivers. continued

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ANIMALS: FISH, SHELLFISH, AND INSECTS 139 BOX 4-1 Continued The use of marine fish cages that are suspended in coastal waters makes it nearly impossible to meet the committee's recommendation to institute integrated confinement systems (Chapter 6). The cages provide weak physical confinement and preclude "end of the pipe" confinement measures such as imposing lethal temperatures or chemical treatment of effluent water through which fish might escape. Thus, the confinement system relies heavily on the biological dimension and hinges specifically on the triploidization success rate. It also depends on the statistical power of detecting fertile diploid fish at different frequencies and sample sizes if culling relies on screening a sample rather than each fish in the lot. A conservative estimate indicates that the cost of screening individual salmon by flow cytometry would add $0.02 to $0.04 per 1 kg of fish to the market cost of farmed Atlantic or chinook salmon (Kapuscinski, 2001). The estimate is consid- ered conservative because it is based on small-scale tests (Wattendorf, 1986), and it does not account for the economies of scale afforded by the use of flow cytometry screening (Harrell and Van Heukelem, 1998). It also does not include the reduced price of labor or the time saved that could be achieved through com- puter automation techniques. In any event, the cost of individual screening is a fraction of the current market price of salmon molts, trout fingerlings, or other early- life stages purchased by grow-out farmers. The second weakness of the proposed bioconfinement is the potential for reproductive interference or other competitive interactions caused by periodic large escapes and possible migration of all-female triploid salmon into rivers. Reproduc- tive interference would occur if the females had reproductive hormone concentra- tions sufficient to cause them to ascend rivers, mate with wild males, and produce infertile broods. Given that males can spawn with more than one female, this would be of greatest concern where most available females were sterile-farm escapees, because the total number of wild adults that return to the rivers would be extremely low in succeeding generations. A lack of appropriate research on the courtship and migratory behavior of triploid all-female salmon makes it difficult to assess the extent to which reproductive interference is a concern. The weaknesses of the proposed bioconfinement measures could be avoided by combining bioconfinement with the much more reliable physical confinement afforded by farming salmon in land-based facilities, ideally in closed-loop recircu- lating aquaculture systems (Kapuscinski, 2003). The salmon farming industry is under increasing pressure to solve a host of environmental problems posed by cage farming regardless of the possible adoption of transgenic salmon. A few entrepreneurs have responded by establishing land-based salmon farms in North America. The initial capital costs and the higher operating costs of land-based operations are major disincentives to an industrywide switch from sea cage to land-based production systems. However, Aqua Bounty Farms has publicly sug- gested that the cost advantage of producing faster-growing transgenic salmon could give salmon farming companies enough economic leeway to make the switch to land-based production (McClure, 2002).

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140 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS production of second-generation tetraploid Pacific oysters (Guo et al., 1996) is stimulating work to establish tetraploid breeding lines that will remove the need to continuously induce tetraploidy. Mosaic individuals: A small percentage of putative triploids can become mosaic--bearing some diploid and some triploid cells--as has been found in studies of fish and oysters (Benfey, 1999; Harrell and Van Heukelem, 1998; Hawkins et al., 1998). Bioconfinement would be compromised if cells within gonadal tissue were mosaic, but no published data were found on searches for this in fish. Research in Pacific oysters has shown that some triploids revert progressively over their lifetime to a mosaic state, raising the possibility that they could produce viable gametes (Calvo et al., 2001; Zhou, 2002). Reductions in triploidy have ranged from 2% to 10% to more than 20% in Pacific oysters (Allen et al., 1996; Nell, 2002). One researcher reported reversion to mosaics to be an order of magnitude lower in interploid triploid oysters (0.6%) than in induced triploids (2.5%10%), although both types had a low incidence of "streakers" that revert to diploidy in all or nearly all tissues (Standish Allen, unpublished data). Variable functional sterility: Even when the induction is successful, the amount of functional sterility achieved is highly variable. Triploidy in fin- fish disrupts gonadal development somewhat in males but more fully in females, with some exceptions (Thorgaard and Allen, 1992). Where triploid females fail to produce viable eggs, combining triploidy with production of all-female lines substantially increases the effectiveness of bioconfinement. Disrupting reproduction in wild relatives: Triploid sterilization would not completely remove the need to assess the ecological consequences of escaped GEOs because triploids of some species have enough sex hormones to cause them to engage in normal courtship and spawning behavior. Escaping triploid fish could interfere with the reproduction of wild relatives by mating with fertile wild adults, leading to losses of entire broods and lowering of reproductive success. The most severe consequence would be reproductive interference in already declining, threatened, or endangered species. Nearly all U.S. salmon populations other than those in Alaska are at risk. There has been little research to investigate the extent to which triploid adults of different fish species retain normal reproductive behavior. In trout and salmon, the concern appears to be mostly with triploid males (Cotter et al., 2000; Inada and Taniguchi, 1991; Kitamura et al., 1991). The risk could be lessened through production of transgenic lines of sterile females (Donaldson and Devlin, 1996). In one of the few field tests of the behavior of triploid fish released into the natural environment, triploid adult Atlantic salmon migrated back from the ocean to natal freshwaters at a much lower rate than did control salmon, thus reducing the population that could attempt to mate with wild fish (Cotter et al., 2000). Virtually

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148 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS For instance, genomic integration of large stacked-gene constructs could increase disruption of favorable endogenous genes, thus imposing a practical limit to the degree of redundancy achievable by this method alone. Weaknesses The blockage of expression of the targeted gene might never reach 100%, raising problems similar to those regarding success rates of triploid induction. Data on effectiveness await completion of development of trans- genic fish lines with stable inheritance and expression of a sterile feral genetic construct (R. Thresher, CSIRO Marine Research, personal commu- nication, May 20, 2003). The blocking of embryonic viability was still well below 100% in early experiments in which the dsRNA was simply injected into whole embryos (Thresher et al., 1999), indicating the need for consid- erable research before this technology can be used for commercial bio- confinement. The expression of the blocker molecule or its promoter could be turned off by methylation (NRC, 2002b) or breakup of the construct during recombination or mutation. This should occur only in a very small fraction of fish that escape from an aquaculture operation or of their fertil- ized embryos, assuming that biotechnologists would have confirmed stable integration, transmission, and expression of the sterile feral construct be- fore commercialization. Natural selection, however, would strongly favor individuals in which the sterility genes failed to express; even a small failure rate among escapees could multiply fairly quickly into a large incidence of fertile transgenic individuals in the wild, especially in cases where the main transgene (not the sterile feral construct) confers some selective advantage over that of untransformed conspecifics. Another potential cause of failure could be the unexpected presence of the repressor molecule, such as tetracycline, in the natural environment in a form and concentration that could successfully repress the lethal sterility genes in a fraction of escaped animals. Elevated concentrations of many biochemicals--antibiotics, caffeine, hormones, and pharmaceuticals--have been recorded in surface water bodies in the United States (Kolpin et al., 2002). Many manufactured biochemicals pass through domestic and indus- trial sewage and stormwater runoff systems in biologically active forms and then enter rivers, lakes, and coastal waters where they can remain in solu- tion in the water column. It then must be determined whether those waters contain a bioactive compound that could repress sterility genes. Should that occur, biotechnologists would have to design a different genetic control system that responds to another compound not present in such quantities.

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ANIMALS: FISH, SHELLFISH, AND INSECTS 149 Default Gene Blocking by Interference RNA and Exogenous Rescue It is possible to incorporate into transgenic constructs sequences that interfere with posttranscriptional expression of a target gene. This general strategy for blocking gene expression uses RNAi and could involve dsRNA (Fire et al., 1998), "hairpin" RNA, or other forms. RNAi would be used to block expression of an endogenous factor that is essential to development or reproduction. That compound would then be supplied exogenously in the diet to allow normal development or reproduction of captive GEOs. Upon escape from confinement the transgenic organisms would not survive or reproduce because necessary substance would no longer be available. Recent advances in silencing diverse target genes in plants (Smith et al., 2000; Wesley et al., 2001; see Chapter 3) open the possibility of developing similar approaches in fish and shellfish: The transfer of intron-containing constructs encoding self-complementary "hairpin" RNA (ihpRNA) led to silencing of the targeted genes in 90100% of individual plants and to a high degree of silencing within individuals; some plants exhibited almost complete knockout of the target gene. For utility in bioconfinement, it would be best to completely silence target gene expression in 100% of the transgenic individuals and to have confirmation of stable inheritance of the intact gene-silencing construct. Strengths Homologous recombination is not necessary for function, only knowl- edge of the sequence of the target genes in the target organisms. Weaknesses Experiments using RNAi in fish have not demonstrated sufficient speci- ficity of target gene inhibition, and RNAi might not be suitable as a mecha- nism for knockdown or knockout of target gene expression . Not enough is known about the effects of this approach on whole fish and shellfish to develop an adequate list of strengths and weaknesses. Ten or more years of research could be needed to reach and verify effective bioconfinement. Externally Administered Gene-Specific Compounds It is possible that various nucleotide analogues could be used as gene- specific compounds to interrupt the expression of developmentally impor- tant genes. Those compounds could prevent development of reproductively necessary organs and gonad tissues, gametes, and other structures in the production organisms (not in the breeding stock) early in development but

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150 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS would not interfere with desirable characteristics of the target organisms. Candidate molecules include nonendogenous analogues of nucleotides that bind to a specific endogenous DNA sequence and interrupt its normal expression (Corey and Abrams, 2001; Ghosh and Iverson, 2000; Heasman, 2002). The targeted binding of those analogues relies on the normal speci- ficity of base pairing, as occurs in the hybridization of naturally occurring nucleic acids. But the analogues have altered properties that are the result of chemical modifications of the backbone structure that supports the nucle- otide bases. The altered backbone makes the analogues resistant to degrada- tion in the target cell, more so than the RNA oligonucleotides involved in the gene-blocking approaches described above. The analogues have been used to shut down or knock down expression of specific genes (a compila- tion of the work using the most successful of these analogues can be found at the Gene Tools web site, http://www.gene-tools.com/). Thus the ana- logues could be adapted to bioconfinement by disrupting development of reproductively essential cells, tissues, or organs without altering desirable characteristics of the target organism. Strengths and Weaknesses This discussion could constitute the first proposal to apply gene ana- logues for bioconfinement, so consideration of strengths and weaknesses is highly speculative at this point. A description of the fundamental advan- tages of morpholino derivatives could give some indication of possible strengths of this general approach. A morpholino is an antisense oligo- nucleotide derived from the morpholine ring, which replaces the ribose or deoxyribose rings characteristic of RNA- and DNA-type oligonucleotides (http://www.gene-tools.com/Questions/body_questions.HTML). Gene Tools currently is the only licensed producer of these compounds in the United States. Mention in this publication does not confer endorsement of the firm or its products). Three general weaknesses would warrant attention if this approach were pursued for bioconfinement. First, the analogues could fail to perform as necessary. The analogues also could be too expensive for widespread use. Finally, the analogues could prove environmentally unstable and thereby present a hazard to nontransgenic organisms. Gene Knockout A target gene could be inactivated by knockout processes similar to those used to produce transgenic knockout mice. However, the process requires the ability to replace the target gene in the target organism with a knockout gene. This is additionally dependent on technology to allow the

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ANIMALS: FISH, SHELLFISH, AND INSECTS 151 production and selection of chimeric progenitor animals through homologous recombination and selection of the desired genotypes. Strengths and Weaknesses This technology is now unavailable or insufficiently robust for applica- tion in a bioconfinement protocol (Rong and Golic, 2000, 2001; Rong et al., 2002). Naturally Sterile Interspecific Hybrids There are few well-documented examples of sterile hybrids among fish and shellfish. All the known examples involve hybrids between taxonomi- cally distinct species, or interspecific hybrids (Chevassus, 1983). One recent study reported highly effective achievement of "natural" sterility through two consecutive but different forms of interspecific hybrid- ization (Liu et al., 2001). The first event, in the F3F8 generations (female red crucian carp male common carp), yielded tetraploids that apparently produce diploid (not haploid) gametes. The second hybridization mated a male F3F8 hybrid (diploid sperm) with haploid eggs from a female of a third species, either Japanese crucian carp or Xingguo red carp. This yielded triploid fish, all of which were sterile. This is an ideal bioconfinement system: It provides 100% sterility of all progeny of the second cross, and it theoretically eliminates the need to screen for failed cases or to bear the added cost of artificially induced triploidy. The challenge is to find similar systems for "natural" bioconfinement across the spectrum of fish and shell- fish species that have suitable characteristics for thriving in aquaculture systems, that consumers are willing to eat, and that aquaculturists are willing to produce. Strengths An interspecific hybrid clearly shown to be 100% sterile but viable and with suitable production characteristics would offer several bioconfinement strengths in production aquaculture: the highest possible reliability for a single confinement measure, ease of application, and obviation of the need for screening to remove potentially fertile individuals as required when relying on triploidization. Weaknesses Given that many interspecific hybrids of fish and shellfish are fertile, it is not safe to assume that any one hybrid is sterile without reliable evidence

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152 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS to the contrary (Thorgaard and Allen, 1992). Also, the degree of sterility in female and male hybrids might not be the same (Donaldson et al., 1993). Combining Triploidization with Interspecific Hybrids Where it is important to prevent reproduction by all individuals, Thorgaard and Allen (1992) proposed the use of interspecific triploid hybrids when the diploid hybrid is either unviable or fertile. Triploid hybrids involving some species of salmon and trout have higher survival rates than do their equivalent diploid hybrids (Chevassus, 1983), and they have been studied to a limited extent (Benfey, 1989). The triploid hybrid might be acceptable to aquaculture producers if it exhibits viability and good perfor- mance in other production traits, such as growth and general resistance to disease. Indeed, the combination of dramatically enhanced production traits in transgenic fish or shellfish with triploid interspecific hybridization to achieve confinement objectives might meet these conditions. Strengths and Weaknesses This approach involves strengths and weaknesses that are similar to those discussed above for sterilization via induction of triploidy. However, concerns about the adverse effects of escapees on wild relatives would apply to either or both parental species, depending on their co-occurrence in accessible ecosystems. Such concern would arise either because some unde- tected percentage of escapees is not functionally sterile or because sterile individuals enter into normal courtship behavior and can therefore disrupt the reproductive success of wild mates. Abandoned and Inappropriate Methods Efforts to render fish or shellfish sterile through surgery or chemical treatment have been abandoned for various reasons. Surgical removal of gonad tissue is the oldest method of sterilizing fish, starting with its use by the Chinese on farmed carp centuries ago and on salmon and trout species from the mid-1700s to the late 1900s (Donaldson et al., 1993). Under experienced hands, surgical sterilization can be effective, and it can offer high recovery rates. But it is not a serious candidate for commercial-scale bioconfinement of transgenic lines because of the cost of labor-intensive surgery and the need to wait until each fish has grown to at least 100g to exhibit gonads. Chemosterilization of fish, through treatment with mutagens, gonadotropin antagonists, antisteriod compounds, and androgens, as well as sterilization by X- or gamma-irradiation, has been abandoned because fish destined for human consumption would pose food safety concerns or

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ANIMALS: FISH, SHELLFISH, AND INSECTS 153 be unacceptable to consumers (Donaldson et al., 1993). In mollusks and crustaceans, chemical shock to induce triploidy, and thus disrupt sexual reproduction, has shown mixed results (Dumas and Campos Ramos, 1999; Fast and Menavesta, 2000). Chemical shocking with cytochalasin B has been highly effective in some species, such as oysters, but it causes high mortality in others. The chemical 6-dimethyl-aminopurine (6-DMAP) appears to be less toxic for inducing triploidy, but it too produces triploidy in fewer than 100% of treated fish. Interspecific hybridization which often fails to disrupt sexual reproduc- tion is relatively common (Collares-Pereira, 1987; Turner, 1984), occur- ring, for instance in at least 56 fish families (Lagler et al., 1977). Because the majority of the known interspecific hybrids of fish and shellfish also are fertile, any new interspecific hybrid combinations that are tried as a bio- confinement measure should be thoroughly screened for evidence of fertility in both sexes. Gene regulation strategies aimed at biological control of pest or nui- sance species are not appropriate for bioconfinement. Consider for example the research under way in Australia to develop transgenic fish lines that bear a "daughterless gene" construct as a strategy for eradication of alien, nuisance fish species that have invaded river systems (CSIRO, 2002; Nowak, 2002; Woody, 2002). The strategy is inappropriate for bioconfinement of transgenic fish and shellfish in aquaculture because the aim is quite the opposite--it is to spread the daughterless gene construct as fully as possible into the alien, nuisance fish population. The general idea would be to release large numbers of alien species fish bearing the daughterless-gene construct among free-roaming individuals of the pest species and thus trigger a collapse of the pest population. BIOCONFINEMENT OF INSECTS As noted in the introduction to this chapter, there are many reasons for producing transgenic insects. It will be important, no matter the justifica- tion, to prevent those insects from going where they are not wanted and to prevent their transgenes from spreading to wild or domesticated populations. Sterile Insect Technique The sterile insect technique (SIT), originally developed for biological control of insect pests, also could be applied to biologically confine trans- genic traits of insects. The traditional approach involves the release of mass-reared and sterilized male insects to mate with wild females, thus reducing the pest population (Braig and Yan, 2002; NRC, 2002b; Wimmer, 2003). Radiation is most commonly applied to colony-reared insects to

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154 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS create mutations that induce sterility. The amount of mutation is adjusted so that every gamete produced by mutagenized insects will contain at least one such lethal mutation. Chemical sterilants were evaluated to induce dominant, developmentally lethal mutations (Borkovec, 1975, 1976; Grover and Agarwal, 1980; Knipling, 1968). However, previously and currently available chemosterilants pose hazards to workers in mass-rearing factories, and available chemicals cannot be applied to the indigenous pest popula- tion without endangering nontarget species. Thus, ionizing radiation, most often from an isotopic source (60Co, 137Ce) or an electron accelerator tuned to produce hard X-rays, is used far more commonly. In SIT for pest control, organisms are grown in a colony and then subjected to treatment that damages their gametes to the point at which no progeny of a mating with the treated insects can survive (Calvitti et al., 1997; Krafsur, 1998). The insects are then released to mate with their wild conspecifics. In the idealized case, all offspring of such mating will receive one copy of a dominant lethal gene. However, population control can be effected with less than absolute sterility. This has been successful in the codling moth Carpocapsa pomonella, where substerilizing doses of radia- tion created males with chromosomal translocations that reduced their fertility. Lucilia cuprina blowfly males were developed with translocations between the autosomes and the Y chromosome (Calvitti et al., 1998; Carpenter et al., 2001; de Azevedo et al., 1968; Gracia and Gonzalez, 1993; Hardee and Laster, 1996; Hasan, 1999; Kerremans and Franz, 1995; Makee and Saour, 1999; Mansour and Krafsur, 1991; McInnis et al., 1994; Qureshi et al., 1993; Seth and Sehgal, 1993). To use SIT for bioconfinement or confinement, sterility should be as close to 100% as possible. However, irradiation or other sterilants may damage the general vigor and competi- tiveness of the treated insects (Stiles et al., 1989). Thus, the use of SIT techniques as a confinement method may conflict with other intended uses, should exposure to sterilants result in a less competitive organism. This must be considered in evaluating SIT technology for bioconfinement of transgenic organisms. For example, should the effective sterilizing dose for a given insect cause a great deal of somatic damage, resulting in a less competitive insect, SIT would not be an effective method. In addition, use of sterilizing technology for bioconfinement would require rigorous quality assurance. Means for ascertaining fertility of insects subsequent to exposure to sterilants of SIT insects do exist, although their successful implementation can depend heavily on species-specific behavior and biology (Katsoyannos et al., 1999; Lux and Gaggl, 1996). Additionally, many of the most effec- tive methods require so much time or such destructive testing of target organisms that it would be unfeasible for a program involving large num- bers of transgenic organisms to be biologically confined. It is more effective

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ANIMALS: FISH, SHELLFISH, AND INSECTS 155 to establish doses of sterilants that cause the desired sterility and then to determine that target organisms receive this dose. Standard methods for the process are available (Committee, 2002). Since its inception, SIT has been applied worldwide to a variety of insects (Table 4-2; Van der Vloedt and Klassen, 1991) indicating that reori- entation of this approach to achieve bioconfinement of transgenic insects (rather than to control pest insects) would be possible for a broad range of species. TABLE 4-2 Insects Subjected to the Sterile Insect Technique Insect 1991 sites Previous sites Screwworm Guatemala, Belize, Libya Curaao, U.S., Mexico, Puerto Rico, U.S. Virgin Islands Mediterranean Guatemala, U.S. (Hawaii) Italy, Peru, Mexico, U.S. fruit fly (California), Israel Caribbean fruit fly U.S. (Florida) fly-free zone U.S. (Florida) Melon fly Japan Oriental fruit fly Japan, Brazil Mariana Islands (Rota), U.S. (Hawaii) Onion fly Netherlands Netherlands control Mexican fruit fly U.S., Mexico U.S., Mexico (quarantine + fly-free zone) Cherry fruit fly Switzerland Tsetse fly (4 species) United Republic of Tanzania, Nigeria, Nigeria, Zanzibar, Burkina Faso Sheep blowfly Australia Tobacco budworm U.S. Stable fly U.S. Virgin Islands (St. Croix) Tsetse fly United Republic of Tanzania, Nigeria, Zanzibar SOURCE: Adapted from Van der Vloedt and Klassan, 1991.

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156 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS The first field release that combined SIT and genetic engineering applied the latter principally to monitor the effectiveness of SIT, but had the corollary effect of confining the transgenes. It involved release of sterile transgenic pink bollworms--a lepidopteran pest of cotton--that bore the marker gene for green fluorescent protein (GFP) as part of a biological control program run by the U.S. Department of Agriculture in the cotton- growing areas of Arizona (Staten et al., 2001). Under ultraviolet light, GFP, even in dead insects, allows visual discrimination of sterile from fertile native bollworms (Braig and Yan, 2002). It should be possible to apply traditional SIT principally to prevent movement of transgenes into wild insect populations, rather than as a biocontrol method for a pest insect. Strengths The techniques developed for pest control that rely on induction of sterility or partial sterility can prevent flow of genetic material into con- specific populations (Marsula and Wissel, 1994; Robinson, 2002). SIT produces infertility through induction of mutation. Ideally, the treatment does not interfere with the desired characteristics of the target organism. Weaknesses Failure of SIT for bioconfinement of transgenic insects in large-scale applications would result from inadequate sterilization in the mass-reared insect population and subsequent release of fertile insects. The rates of sterility, in terms of fertile offspring of steriles in practice, vary from effec- tively 100% to 75%, depending on the target organism. Sterility in Dipteran flies is usually high, effectively 100%, whereas in other insects the sterility can be lower and still be effective in pest control. Thus the use of SIT in bioconfinement must consider the response of the target organism to the sterilizing method. Transgenic Sterile Insects Gene transfer also has been proposed as a way to produce sterile insects for biological control that would improve on the traditional SIT approach and replace the use of radiation or other mutagens to induce sterility (Alphey, 2002; Alphey and Andreasen, 2002; Thomas et al., 2000; Wimmer, 2003). An important motivator for this line of research is that radiation- based SIT tends to depress the vigor and competitive ability of sterile males, thus undermining SIT's effectiveness for biological control. This also will be a concern if SIT approaches are applied to biologically confine transgenic

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ANIMALS: FISH, SHELLFISH, AND INSECTS 157 insects. Ideally, bioconfinement methods would abrogate reproduction with- out altering any other desirable traits. One strategy involves developing transgenic traits for inducible genetic sterility. An example demonstrated in fruit flies involves a transgene-based dominant embryonic lethality system that can generate large quantities of competitive but sterile insects (Horn and Wimmer, 2003). The sterile insects are vigorous adults but their transgenes cause lethality after transmission to progeny. This embryonic lethality can be suppressed maternally in the labo- ratory in order to propagate the strains. Transgene-based embryonic lethality can combine with another strat- egy involving transgenic female-specific lethality systems to produce sterile males (Heinrich and Scott, 2000; Thomas et al., 2000). Female-specific lethality can be turned on and off through inclusion of a tetracycline- activated regulatory element in the transgenic construct. The construct can be suppressed by supplementing food with tetracycline during insect rearing in captivity. Strengths and Weaknesses Almost nothing is known about the strengths or weaknesses of these transgenic methods for bioconfinement because scientists are at least 10 years away from application. As has been the case for traditional SIT, the rates of sterility, in terms of fertile offspring of steriles, will likely vary from effec- tively 100% to 75% depending on the target organism and the specific transgenic method used. Ecological Characteristics of Production Site For commercially important and partly or wholly domesticated trans- genic insects, the amount of confinement needed depends strongly on the insect's biology. In the case of silkworms, little or no confinement should be necessary because the insects are completely adapted to commercial silk production, so they cannot escape. However, low vagility (mobility) cannot be expected should transgenic honeybees be produced, because of the possibility that transgenic bees would mate with wild-type bees of the same species. Climatic or ecological conditions in some places should provide con- finement for transgenic insects, depending on the insect's ecology and behavior and on the feasibility of keeping it confined to that region. How- ever, inadvertent or purposeful transport to a more suitable area could easily abrogate such confinement. For example, the Mediterranean fruit fly (medfly) and other tropical insects would have no chance of survival in the

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158 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS immediately accessible environment should they escape from a rearing facility located in an area with a cold climate or lacking appropriate hosts. Although the diverse diet of the medfly makes this latter confinement approach problematic, it could be implemented where the insect in question has a highly restricted host range. Fitness Reduction and Regulation of Gene Expression Some transgenic, mass-reared insects that serve as biological factories to produce valuable proteins could escape confinement and interbreed with their wild specifics. For instance, medflies and pink bollworms can be engineered to produce valuable transgenic proteins (Peloquin and Miller, 2000). Although it is unlikely that a medically or industrially important protein produced by such a transgenic insect would confer any selective advantage, it is not, at the very least, good environmental hygiene to allow the escape of such a transgenic insect. Perhaps such biological factory insects could be rendered flightless or incapable of long-range dispersal by use of a flight-defective mutation, such as the long-known recessive Drosophila gene vg, which results in flightless insects. Alternatively, technology for gene blocking or gene knockout in development for bioconfinement of transgenic fish and shellfish might be developed to prevent reproduction or postescape survival of industrial transgenic insects.