Available online at www.sciencedirect.com
Comparative Immunology, Microbiology
and Infectious Diseases 32 (2009) 139–161
www.elsevier.com/locate/cimid
Transgenic fish resistant to infectious diseases,
their risk and prevention of escape into the
environment and future candidate genes
for disease transgene manipulation
Rex A. Dunham *
Department of Fisheries and Allied Aquacultures, Auburn University, Alabama 36849, USA
Abstract
Transgenic fish have been produced that have improved growth, disease resistance, survival in
cold and body composition, have altered color, that can act as bioindicators for estrogenic
pollutants and that can produce pharmaceutical proteins. The largest amount of transgenic
research has focused on growth hormone transfer. A relatively small amount of research has
focused on enhancing disease resistance, but significant enhancement has been accomplished.
Pleiotropic effects from the transfer of other transgenes, particularly growth hormone gene
can alter disease resistance in both positive and negative ways. Most negative effects
for all transgenes appear to lower fitness traits, which is positive for biological containment.
Transgenic fish appear to pose little environmental risk, but this research is not fully conclusive.
To expedite commercialization and minimize environmental risk, transgenic sterilization
research is underway. A large amount of functional genomics research has resulted in a much
better understanding of gene expression when fish are experiencing disease epizootics. This
information may allow the future design of more effective transgenic approaches to address
disease resistance.
# 2008 Elsevier Ltd. All rights reserved.
Keywords: Transgenic fish; Pleiotropic effects; Disease resistance; Gene expression; Environment; Sterilization
* Fax: +1 334 844 9208.
E-mail address: [email protected].
0147-9571/$ – see front matter # 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cimid.2007.11.006
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R.A. Dunham / Comp. Immun. Microbiol. Infect. Dis. 32 (2009) 139–161
Résumé
Des poissons transgéniques ont été préparés pour améliorer leur croissance, leur résistance aux
maladies, leur survie dans le froid et leur composition corporelle, pour modifier leur couleur et pour
être utilisés comme bioindicateurs permettant de détecter des polluants oestrogéniques ainsi que pour
produire des protéines d’intérêt pharmaceutique. La plus grande partie des études appliquées mettant
en œuvre des poissons transgéniques s’est focalisée sur le transfert du gène de l’hormone de
croissance. Une partie plus modeste des recherches porte sur la lutte contre les maladies, qui ont
permis des progrès substantiels. Des effets pléiotropiques résultant du transfert d’autres gènes et en
particulier du gène d’hormone de croissance, peuvent modifier la résistance aux maladies dans un
sens positif ou négatif. La plupart des effets négatifs de tous les transgènes semble diminuer l’aptitude
des poissons à s’adapter au milieu sauvage ce qui contribue à renforcer l’efficacité du confinement
biologique. Les poissons transgéniques semblent poser peu de problèmes environnementaux mais les
recherches dans ce domaine ne sont pas pleinement concluantes. Pour accélérer la commercialisation
des poissons transgéniques et minimiser les risques environnementaux, des recherches sont actuellement conduites dans le but de stériliser les animaux. De nombreuses études de génomique
fonctionnelle ont permis de mieux comprendre l’expression des gènes quand les poissons souffrent
de maladies épizootiques. Ces informations permettent d’envisager des protocoles nouveaux basés
sur des approches transgéniques plus efficaces pour aborder la résistance aux maladies.
# 2008 Elsevier Ltd. All rights reserved.
Mots clés : Poisons transgéniques ; Effets pléiotropiques ; Résistance aux maladies ; Expression de gènes ;
Environnement ; Stérilisation
1. Introduction
One of the greatest future potential benefits of gene transfer in fish will be enhancement
of disease resistance. Diseases are the greatest problem facing aquaculture and damaging
its profitability. Disease resistance is also an animal welfare issue. Transgenic fish with
enhanced disease resistance would increase profitability, production, efficiency and the
welfare of the cultured fish.
Research to date indicates great promise for success of this approach for enhancing
disease resistance. Genetic gain is also possible through traditional selective breeding, but
it appears that the rate of genetic improvement and the consistency of genetic improvement
may be greater with the transgenic approach [1,2]. Selective breeding may also have the
drawback that the disease organisms may well respond to selective forces as well, negating
some of the selection response in the fish.
2. Trangenesis for disease resistance
2.1. Viral diseases
Anderson et al. [3] provided the first evidence of the potential for transgenic enhancement
of a fish resistance when they used the expression of viral coat protein genes or antisense of
viral early genes to improve viral resistance in rainbow trout (Oncorhynchus mykiss). Shrimp
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141
have been genetically engineered with antisense Taura syndrome virus-coat protein gene [4].
When challenged with Taura virus transgenic shrimp has 83% survival and controls had 44%
survival. More research is needed to use these approaches to combat viral diseases of fish and
shellfish.
2.2. Bacterial diseases
Bacterial disease resistance may be easier to genetically engineer than for diseases
caused by other classification of pathogens, and is better studied. Bacterial disease
resistance may be improved up to three to fourfold through gene transfer. One approach
that has been utilized is the transfer of antibacterial peptide genes.
Immunity conferred on fish by transgenically encoded antibacterial peptides has several
potential advantages over conventional immunization. First, the fish will in principle be
protected (by the expressed peptide) from early in development, long before the immune
system has matured to a stage where specific immunity can be elicited by immunization.
Second, immunity conferred by a transgene removes the necessity for preparing vaccines
for any pathogen against which the peptide is active. These advantages should, if realized,
result in innately disease-resistant fish which would not require the expense or labor of
specific immunization for certain pathogens, and thus offer economic potential for
aquaculture industries.
Insertion of the lytic peptide, cecropin B construct driven by the CMV promoter
enhanced resistance to bacterial diseases such as columnaris and enteric septicemia of
catfish two- to fourfold in channel catfish, Ictalurus punctatus [2]. Cecropins, are small
cationic peptides found originally in the moth Hyalophora cecropia [5] with broad
spectrum antibacterial properties against Gram-negative bacteria [6]. A greater percentage
(100%) of transgenic individuals containing preprocecropin B construct survived than nontransgenic controls (27.3%) during an epizootic of Flavobacterium columnare in an
earthen pond. In this case, the transgene appears to have imparted complete resistance, and
the transgenics had a 3.66-fold higher survival than the controls.
Also, a greater percentage (40.7%) of transgenic individuals containing cecropin B
construct survived than non-transgenic controls (14.8%) when challenged with
Edwardsiella ictaluri, causative agent of enteric septicemia of catfish, ESC, in tanks.
There were no pleiotropic effects, and growth rate of the transgenic and non-transgenic
siblings was not different.
These results also suggest that type of construct may be quite important. The more
natural preprocecropin B appeared to impact greater disease resistance than the cecropin,
which had an artificial catfish leader. However, the results were generated utilizing two
different pathogens so direct comparison of the results is partially flawed.
An alternative to gene transfer for genetic enhancement of disease resistance is selection.
Ehlinger [7] reported that selected strains of brown trout, Salmo trutta, demonstrated a 92%
survival rate while the control groups exhibited a 10% survival when exposed to furunculosis.
Amend and Nelson [8] reported differences in sockeye salmon progeny that resulted from
brood stock selected for resistance to infectious hematopoietic necrosis (IHN).
In the case of channel catfish the results have been variable for selection for increased
disease resistance. Kansas strain channel catfish, I. punctatus were challenged for
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resistance to E. ictaluri with no response to selection [9]. Waters [10] measured response to
selection and correlated response to selection for resistance to ESC and columnaris in
channel catfish. Response to selection for resistance to disease calculated as percent gain
ranged from 2.9% to 128% for columnaris and 97.0% to 26.0% for ESC. Correlated
response to selection ranged from 40.8% to 32% for columnaris and 18.7% to 93.8%
for ESC. A positive response was obtained for 2 of 3, and 1 of 2 lines to selection for
resistance to columnaris, and resistance to ESC, respectively. A positive correlated
response was obtained for 1 of 5, and 2 of 3 lines to selection for resistance to columnaris,
and resistance to ESC, respectively. Direct selection for disease resistance was slightly
more successful than indirect selection for disease resistance. The transgenic approach
appears to have better potential for improving resistance to bacterial pathogens in channel
catfish as the magnitude and consistency of the improvement is better via transgenesis.
Transfer of cecropin genes to medaka has also imparted enhanced disease resistance
[11]. F2 transgenic medaka from different families and controls were challenged with
Psuedomonas fluorescens and Vibrio anguillarum which killed about 40% of the control
fish in both cases, but only 0–10% of the F2 transgenic fish were killed by P. fluorescens and
about 10–30% killed by V. anguillarum. When challenged with P. fluorescens, zero
mortality was found in one transgenic fish family carrying preprocecropin B and two
families with porcine cecropin P1, whereas 0–10% cumulative mortality was observed for
five transgenic families with procecropin B and two families with cecropin B. This is
important as family variation can be extreme for transgenic fish potentially because of
differences in genetic background, variable insertions sites, copy number, epistasis and
other factors. This necessitates coupling selection with gene transfer to obtain maximum
genetic gain from the gene transfer.
When challenged with V. anguillarum, the cumulative mortality was 40% for nontransgenic control medaka, 20% in one transgenic family carrying preprocecropin B,
between 20% and 30% in three transgenic families with procecropin B and 10% in one
family with porcine cecropin P1. Cecropin has also shown anti-viral properties in vitro.
Chiou et al. [12] examined in vitro effectiveness of native cecropin B and a synthetic
analog, CF17, for killing several fish viral pathogens, infectious hematopoietic necrosis
virus (IHNV), viral hemorrhagic septicemia virus (VHSV), snakehead rhabdovirus
(SHRV), and infectious pancreatic necrosis virus (IPNV). When these peptides and viruses
were co-incubated, the viral titers yielded in fish cells were reduced from several- to 104fold. Transgenic rainbow trout containing a synthetic cecropin construct exhibited
increased viral resistance (Thomas Chen, personal communication).
Grass carp, Ctenopharyngodon idellus, have been transfected with carp B actin- human
lactoferrin gene. P1 individuals were more resistant to Aeromonas, exhibited enhanced
phagocytosis and more viral resistance than controls [13].
Yazawa et al. [14] linked Japanese flounder keratin promoter to both the hen egg white
(HEW) lyoszyme gene and green fluorescence protein (GFP) gene, and then produced F2
transgenic zebrafish, GFP expression was strong in the epithelial tissues, liver and gill in all
life stages. Expression of HEW lysozyme was observed in the liver and skin. HEW
lysozyme and GFP were present in the liver of transgenic zebrafish, but not in the muscle.
The lytic activity of protein extracts from the liver was 1.75 times higher in F2 transgenic
zebrafish than in the control. In a challenge experiment, 65% of the F2 transgenic fish
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143
survived an infection of F. columnare and 60% survived an infection of Edwardsiella tarda,
whereas 100% of the control fish were killed by both pathogens.
To date, two types of transgenic processes have been successful for improving diseases
resistance, blocking viruses with antisense and overexpressing antibacterial compounds
from distant taxa. This is the extent of reported research using transgenesis to directly
improve disease resistance. However, transfer of other genes can indirectly affect disease
resistance through pleiotropy both in a positive and negative way. If the pleiotropic effects
are known, they could be intentionally manipulated for genetic gain in a manner analogous
to indirect selection.
3. Pleiotropic effects
The insertion of a transgene, a single gene, can affect more than one trait, pleiotropic
effects. If the breeder is fortunate, these pleiotropic effects will impact other traits in a
positive way.
Survival is an important commercial trait, and the insertion of the rtGH gene altered the
survival of common carp [15]. F2 progeny inheriting this transgene had higher survival
than controls when exposed to a series of stressors and pathogens such as low oxygen,
anchor worms, Lernia, Aeromonas and dropsy. Wang et al. [16] produced results that may
explain the observed increased disease resistance of the GH transgenic common carp in this
earlier study. As expected, F2 ‘‘all-fish’’ growth hormone transgenic common carp had a
mean body weight, 63.4 (6.65) g, higher than that of the controls 39.2 (3.30) g.
Transgenic individuals had higher lysozyme activity 145.0 (30.7) U/ml in the serum
compared to 105.0 (38.7) U/ml for age-matched non-transgenic control fish serum. The
serum bactericidal activity in the transgenics was 18.7% higher than that in non-transgenics
as percentage serum killing was 59.5% (6.83%) and 50.8% (8.67%), respectively.
Values for leukocrit and phagocytic percent of macrophages in head kidney were higher in
transgenics than controls but, the phagocytic indices in the transgenics and the controls
were not different. There was no difference in the relative weight of spleen between the
transgenics and the controls, with the spleen mass index being 0.21% (0.02%) and 0.20%
(0.03%), respectively. GH transgene expression apparently not only stimulated growth,
but also the non-specific immune functions of common carp.
Conversely, GH transgenic salmon were more sensitive to Vibrio compared to controls
[17]. Survival among GH salmon families was sometimes improved, sometimes decreased
and sometimes unchanged relative to controls [18]. These differences in salmon could be
related to alterations in expression for a myriad of disease related genes in relation to the
altered growth hormone expression. Growth hormone appears to have pleiotropic effects
and causes a cascade of events in a large number of biochemical pathways. Growth
hormone (GH) transgenic amago salmon (Oncorhynchus masou) containing the sockeye
salmon GH1 gene fused to the metallothionein-B (MT-B) promoter from the same species
increased body weight approximately four to five times more than control salmon in F2 and
F3 generations [19]. Heme oxygenase, Acyl-CoA binding protein, NADH dehydrogenase,
mannose binding lectin-associated serine protease, hemopexin-like protein, leucytederived chemotaxin2 (LECT2), and many other genes had enhanced expression in hepatic
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tissue of immature transgenic salmon while, complement C3-1, lectin, rabin, alcohol
dehydrogenase, Tc1-like transposase, and pentraxin genes had decreased expression
compared to non-transgenic controls. Gene expression pattern changed when transgenic
salmon approached maturation with hemopexin-like protein, heme-oxygenase, inter alpha
-trypsin inhibitor, LECT2, GTP cyclohydrolase I feedback regulatory protein (GFRP), and
bikunin having enhanced expression and lectin, apolipoprotein, and pentraxin exhibiting
depressed expression. In particular, lectin was found to be highly suppressed in all F2 and
immature F3 salmon. Serum lysozyme activity of the innate immunity system, was
decreased in both generations of GH transgenic fish. GH transgenic amago salmon had
altered hepatic gene expression relating to iron-metabolism and innate immunity.
GH gene transfer affects respiration which in turn could have a multitude of intertwined
relationships with and effects on growth, low oxygen tolerance, disease resistance, stamina
and predator avoidance. When subjected to low dissolved oxygen, 0.4 ppm, mean absolute
survival was the same for transgenic rtGH and control common carp [15,20]. However,
when mean survival time was calculated, the transgenic individuals had longer mean
survival time than the non-transgenic full-siblings. Ventilation rate could also be a possible
explanation for the slightly better tolerance of low oxygen exhibited by the transgenic
common carp. Transgenic channel catfish with the same rtGH construct as the common
carp had a lower ventilation rate when subjected to low dissolved oxygen compared to
controls (Dunham, unpublished data).
Pleiotropy of GH gene for oxygen tolerance characteristics varies from one species to
another. GH tilapia [21] have a 58% higher metabolism than controls, compensate for
oxygen consumption and have the same maximum swim speed as non-transgenics. GH
tilapia tolerate hypoxia equally as well as controls despite higher demand for oxygen.
However, GH transgenic salmon have an increased need for dissolved oxygen [22–24].
However, after 4 days of starvation, GH individuals had the same oxygen uptake as controls
[25]. After feeding, GH transgenics had 1.4–1.7-fold more O2 demand even when the
controls consume equivalent amounts of feed. Adult transgenics had higher oxygen
demand, poorer swimming ability and longer recovery time compared to ocean ranched
salmon [26].
Deitch et al. [27] examined the cardiorespiratory physiology of size-matched GH
Atlantic salmon (Salmo salar). The GH transgenic salmon had a 3.6 faster growth rate,
and 21% and 25% increases in mass-specific routine and standard oxygen consumption
(M ðO2 Þ ), respectively, but no simultaneous increase in their maximum M ðO2 Þ . This resulted
in an 18% lower metabolic scope and a 9% reduction in critical swimming speed. This
decreased metabolic capacity was unexpected given that the transgenics had a 29% larger
heart with an 18% greater mass-specific maximum in situ cardiac output, a 14% greater
post-stress blood haemoglobin concentration, 5–10% higher red muscle and heart aerobic
enzyme (citrate synthase or cytochrome oxidase) activities, and twofold higher resting and
1.7 higher post-stress, catecholamine levels. Gill surface area was the only
cardiorespiratory parameter that was not enhanced, and gill oxygen transfer may have
been the limiting respiratory factor.
There were significant metabolic costs associated with GH transgenesis in this line of
Atlantic salmon. Cardiac function was enhanced by GH transgenesis. Universal
upregulation of post-smolt (adult) GH transgenic salmon cardiorespiratory physiology,
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145
as suggested by symmorphosis, did not occur. Differences in arterial oxygen transport such
as cardiac output and blood oxygen carrying capacity are important determinants of
differences in aerobicity, however diffusion-limited processes may be bottlenecks that
would need to be enhanced to achieve substantial improvements in metabolic and
swimming performance. These diffusion related limiting factors associated with gill
function and morphology may explain differences in results from one study to another.
GH gene transfer alters respiration and metabolism in many ways, which could affect
the ability to overcome diseases either in an enhanced or detrimental manner. The results
from common carp indicate GH transfer could be used as an indirect method to
transgenically enhance disease resistance. The salmon situation may be different because
of their different life history, the fact that they are a cold rather than warm water fish and
their GH enhancement is much more dramatic. The extent of pleiotropic effects is likely a
product of the magnitude of the change in the primary target trait and the associated
expression strength of the transgene.
4. Genotype–environment interactions
The best genotype for one set of environmental circumstances is not necessarily the best
genotype for a second set of environmental circumstances. Genotype–environment
interactions occur either when the value of the genotypes change in rank or the relative
value of two genotypes substantially change in relation to each other [28].
When challenged with either F. columnare or E. tarda, 65% and 60% of the F2
transgenic zebrafish having Japanese flounder keratin promoter-hen egg white lysozyme
fusion gene survived, respectively, whereas 100% of the control fish were killed by both
pathogens [14]. However, a genotype environment interaction occurred as the survival
rates of the transgenic fish were not significantly higher than controls when higher
concentrations of bacteria were used resulting in a more severe challenge.
Protocols for conducting challenge experiments can lead to genotype–environment
interactions, affecting the outcome and conclusions regarding evaluation of fish transgenic
for disease resistance. This must be considered for design of experiments to develop
transgenes for disease resistance, and there is a need to evaluate and contrast results from
directed challenges and from more natural epizootics.
5. Gene expression and future candidate genes for transgenic enhancement of
disease resistance
A tremendous amount of research on functional genomics of disease resistance in fish
has been accomplished. Identification of key gene expression during response to attack by
pathogens may allow identification of important genes for possible transgenic
manipulation.
The acute phase response (APR) is one of the critical first steps of defense against
disease caused by pathogens. The liver is a central site for the acute phase response
component of innate immunity [29], and acute phase proteins (APP) [30] are known to play
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beneficial roles in mediating the complex inflammatory response and restoring
homeostasis following infection or injury [31]. In general, the liver is an important
organ for gene expression related to the immune response in fish [32,33]. A large number of
cytokines, complement components, pathogen recognition receptors (PRR), and
antimicrobial peptides from several aquaculture species have been discovered and
reviewed [34].
In the case of channel catfish, 35 of the 127 upregulated genes in response to exposure to
ESC were acute phase proteins [35], including coagulation factors, proteinase inhibitors,
transport proteins, and complement components. Particularly high up-regulation (>50fold) occurred for genes involved in iron homeostasis (intelectin, hemopexin, haptoglobin,
ferritin, and transferrin). Up-regulation of the majority of the complement cascade
including the membrane attack complex components and complement inhibitors was also
observed. A number of pathogen recognition receptors (PRRs) and chemokines were also
differentially expressed in the liver following infection and chemokines appear to have a
very important role in ESC resistance.
Similarly, in blue catfish [29] at least 20 of the 98 upregulated genes represented acute
phase proteins, including the acute phase response, complement activation and metal ion
binding/transport categories, and were among the most highly upregulated transcripts
following ESC infection. An active complement response to infection was observed, with
three forms of complement C3 upregulated along with C4 and members of the membrane
attack complex (C7 and C9). The complement regulatory protein factor H was also
strongly upregulated (>14-fold). Similar to channel catfish, genes involved in iron binding
and transport were strongly induced following infection including intelectin, haptoglobin,
hemopexin/warm–temperature–acclimation-related 65 Kd protein, ceruloplasmin, and
transferrin. Additional upregulated APP included pentraxin (serum amyloid P-like),
fibrinogen, and angiotensinogen. Upregulated genes involved in immunosurveillance,
immune signaling, and immune cell activation were observed. Other major categories of
upregulated genes have the following functions: acute phase response; complement
activation; metal ion binding/transport; immune/defense response; protein processing,
localization, folding; and protein degradation.
In the case of Japanese flounder, Paralichthys olivaceus, VHSV G gene elicited strong
humoral and cellular immune responses probably protecting the fish during infection from
viral hemorrhagic septicemia (VHS) [36]. Non-specific immune up-regulation was
observed from genes such as NK, Kupffer cell receptor, MIP1-alpha and Mx1 protein gene,
and specific immune-related genes such as the CD20 receptor, CD8 alpha chain, CD40 and
B lymphocyte cell adhesion molecule.
When channel catfish and blue catfish are under attack from bacteria, a large number of
genes are upregulated in the liver, but very few are down regulated [29,35]. Upregulation of
genes in response to disease challenge in channel catfish was 207, and was 5 for
downregulation, and for blue catfish, 98 upregulated and 5 down regulated genes in the
liver after infection with Gram negative bacterium E. ictaluri. Three catfish genes down
regulated were selenoproteins P1b and selenoprotein H which may possess antioxidant
properties [37] in addition to a cell cycle gene, anaphase promoting complex subunit 13
[29]. Similarly, in channel catfish only a few genes including antimicrobial peptide-2, and
thioredoxin-interacting protein [38] which functions in the oxidative stress response in
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147
mammals were significantly downregulated following infection. Also in salmon, a much
smaller number of transcripts were downregulated than upregulated, a seemingly
characteristic result of transcriptomic analyses of bacterial infections [33].
However, Atlantic salmon challenged with the bacterium, Piscirickettsia salmonis, had
71 transcripts upregulated and 31 different transcripts downregulated in macrophages, and
30 different transcripts upregulated and 39 different transcripts downregulated in
hematopoietic kidney [39]. The relationship of number and proportion of up and
downregulated genes in response to attack by pathogens may vary by tissue, cell type,
species of fish and pathogen, and needs to be determined.
Fish have some unique, unidentified disease resistance genes yet to be fully described
and understood. Several unidentified genes were upregulated and induced by viruses,
bacterial endotoxins or mitogen in Japanese flounder leukocytes [40]. Significant up- and/
or downregulation of unknown genes was observed. Atlantic salmon challenged with the
bacteria, Aeromonas salmonicida, had differential gene expression for humoral
components of the innate immune system, genes not previously associated with infection,
and a number of genes with no known homologs [33]. These numerous transcripts that are
differentially expressed in fish during infection that are unidentified could represent a
subset of genes that respond to infectious agents that do not have this role in or could be
absent from warm-blooded animals. Eighty such differentially expressed genes respond to
bacterial exposure in channel catfish and 20 in blue catfish [29,35].
Some genes seem to be key for disease resistance. As previously mentioned, iron
homeostasis, binding and transport in channel catfish appears to be a critical immune
response [35]. These transcripts included intelectin, the most highly upregulated gene
observed at >85-fold, haptoglobin (>34-fold), hemopexin (>25-fold), ceruloplasmin (8.5fold), transferrin (>7-fold), and ferritin (>2-fold). Intelectin was also the most highly
upregulated gene in ESC infected blue catfish, 455-fold [29]. Intelectin was also
upregulated in carp [41] and rainbow trout [42]. In mammals, intelectin is believed to be
involved in pathogen defense mechanisms, recognizing galactofuranose in carbohydrate
chains of bacterial cell walls [43] and may function as a receptor for lactoferrin, an iron
sequestering homologue of transferrin [44].
Chemokines also appear to be key disease resistance genes in catfish. Two blue catfish
CC chemokines, SCYA106 and SCYA113, previously identified from catfish were highly
induced [38,45]. SCYA106 was upregulated >105-fold, and based on comparative
mammalian data [29,46] SCYA106 and SCYA113 may be regulators of dendritic cell
trafficking to secondary lymphoid organs. Upregulation of CCL19-like genes after
infection has also been recently reported in rainbow trout and Atlantic salmon [32,47]. A
catfish orthologue of CXCL14 chemokine [48] also exhibited elevated expression in the
liver after infection, which in mammals, acts as a chemoattractant for activated monocytes,
immature dendritic cells, and NK cells [49].
Fish also possess antibacterial peptides/genes. Oddly, the channel catfish liverexpressed antimicrobial peptide-2 [38] was downregulated in response to ESC infection
[35]. This peptide should be in increased in expression if it is to battle invading bacteria.
Japanese flounder have two lysozyme, single copy genes, (muramidase) genes c- and gtype [50]. Their gene products were able to lyse V. anguillarum and Pasteurella piscida, but
not the two major pathogens of Japanese flounder, E. tarda and Beta streptococcus. E.
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tarda induced lysozyme production even though the lysozyme was not effective in lysing
this bacterium. It seems logical that the most devastating pathogens for a species would be
ones that have evolved to circumvent the natural immune and defense mechanisms. In this
case the invader induces the counterattack, but somehow has developed its own defense
mechanism. Likewise, long-term selection for disease resistance may never achieve total
resistance as the pathogen counters with its own selection response, and the variety of
strains of pathogens within a single species of pathogen may allow circumvention of host
defenses, and result in varying heritabilities dependant on the serotype used for the
challenges. If this is true, gene transfer of constructs from distantly related species or
different taxa may be more effective in genetically improving disease resistance than
transgenically manipulating genes intraspecifically. Here we have two examples where the
native antibacterial peptides had little effect on disease resistance, but transgenes from
completely different taxa, insects, birds, mammals and synthetics were very effective in
imparting disease resistance.
The catfish bacterial challenges showed that acquired immunity genes also responded to
bacterial, E. ictaluri, invasion [29] and, a large number of [35] transcripts with functions in
protein modifications and degradation were upregulated in the liver of catfish following
infection. Members of these two groups of genes were likely connected to the endoplasmic
reticulum’s (ER) unfolded protein response (UPR) which upregulates chaperones and
genes for protein degradation upon the accumulation of unfolded proteins during stress
[51], or to the degradation and processing of antigens for the MHC class I molecule [29]. At
minimum, 15 unique transcripts were upregulated in these two categories including
chaperones, proteasome activators, and proteasome subunits.
The upregulation of two different MHC class I alpha chains and beta-2-microglobulin
(b2m) indicated active antigen processing and presentation were likely occurring in the
blue catfish liver after infection with E. ictaluri, an intracellular bacterium, as part of a cellmediated immune response [35]. Similar to what is seen in mammals, genes associated
with the generation of peptides and peptide-loading for the MHC class I molecules, PA28 a
and b, were upregulated in blue catfish liver. Both PA28 a and b proteasome activator
subunits were upregulated in blue catfish, suggesting a shift toward MHC class I antigen
processing. Two ER chaperones, calreticulin and endoplasmin (GRP94), were also
induced, providing further evidence of an active MHC class I-mediated response as well as
tapasin (2.3-fold), another molecule involved in MHC class I antigen loading. The
coordinated upregulation of MHC class I alpha chain, b2m, and PA28-b has also been
reported in large yellow croaker (Pseudosciana crocea) following poly I:C injection [52].
The example provided by blue catfish and channel catfish shows the complexity in
choosing candidate genes for transgene manipulation, and this will impact the schemes and
designs needed for gene manipulation in the future as their gene expression in response to
ESC was significantly different. Both species shared a wide spectrum of similarities in gene
expression profiles after infection including an acute phase response, and strong induction
of complement components and iron regulatory genes at day 3 after infection [29,35].
However, a total of 58 genes were differentially expressed in blue catfish liver, but not in
channel catfish liver at day 3 after infection. CC chemokine SCYA106, the most highly
induced transcript in blue catfish, was not differentially expressed in channel catfish.
Several MHC class I-related components, as well as several other immune-related genes,
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149
were upregulated in blue catfish but not in channel catfish. A few large expression
differences between blue and channel catfish were observed at 24 h after infection, and
there was little evidence of early induction of these genes in channel catfish. However,
matrix metalloproteinase 13 (MMP-13), was upregulated more than 20-fold in channel
catfish at 24 h with only a slight induction of expression relative to blue catfish. This gene is
also induced following E. tarda infection in Japanese flounder [53].
Perhaps particular attention to expression patterns in moribund fish should be
considered when choosing candidate genes. Induction of expression of the studied genes
was generally higher in moribund catfish [29,35]. Expression of SCYA106 and lysosomalassociated membrane protein 3 (LAMP3) rose sharply in both dying blue catfish and
channel catfish relative to controls, but showed greater upregulation in blue catfish. MMP13 induction in moribund channel catfish was markedly higher than that observed in
moribund blue catfish.
The induction of genes involved in MHC class I cascades in blue catfish was a major
difference between blue catfish and channel catfish expression profiles [29,35]. An earlier,
or more efficient, MHC class I/CTL response to the intracellular bacteria could potentially
account for some of the phenotypic differences in resistance in the two species, blue catfish
being more resistant. MHC class I-related components including MHC class I alpha chain,
beta-2-microglobulin, and proteasome activator PA28 alpha genes showed little induction
in channel catfish or blue catfish at 24 h but were strongly upregulated in moribund fish of
both species. MHC class I components were also upregulated in channel catfish, but at a
later time point than in blue catfish. Similarly, CC chemokine SCYA106 expression, while
little changed in either species at 24 h, was drastically induced in moribund fish.
Additionally, channel and blue catfish chemokine have 33 different chemokine genes
[54]. Species differences in gene expression when challenged with ESC were also observed
in the kidney as well as the liver. After ESC infection dramatic up-regulation up to 200-fold
was found for channel catfish, which is susceptible to ESC, CXCL10 chemokine in head
kidney, while blue catfish, which is resistant to ESC, CXCL10 showed a modest induction
24 h after infection In the case of channel catfish CXCL8 chemokine, expression increased
about three to fivefold at 24 h after infection, while the upregulated expression in the blue
catfish was detected at 72 h after infection.
Gene expression can vary among cell types and tissues. In Atlantic salmon, ten
antioxidant genes were upregulated in infected macrophages, but not in infected
hematopoietic kidney [39]. Transcripts of the adaptive immune responses such as T cell
receptor alpha-chain and C–C chemokine receptor 7 were downregulated in infected
hematopoietic kidney, but not in infected macrophages may be associated with infectioninduced kidney tissue damage.
There appear to be several generalizations among fish and mammals in regards to gene
expression for disease resistance. The blue catfish and channel catfish APR as measured
three days after infection [29,35] included many of the components of the typical
mammalian APR and also contained commonalities with APR in salmonids and carp
[32,33,41,42,55]. Acute phase proteins such as haptoglobin, hemopexin, transferrin,
ceruloplasmin, fibrinogen, angiotensinogen, pentraxin and several complement components accounted for a significant percentage of upregulated transcripts in blue catfish [29],
in rainbow trout [42] and zebrafish [56], indicating the likely conservation of function of
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the vast majority of APP between mammals and teleost fish. Pentraxin, upregulated 4.1fold in blue catfish [29], initiates the complement cascade and possesses opsonizing
activity in the snapper Pagrus auratus [57,58]. The complement system of teleost fish plays
conserved roles in sensing and clearing pathogens [59] as indicated by the presence of C3,
the central component of the complement system, in multiple forms in fish, possibly
serving as an expanded pathogen recognition mechanism [60]. Three forms of C3
important for the activation of the lectin and classical complement pathways, and two
components of the membrane attack complex which carries out cell lysis, C7 and C9, were
upregulated in blue catfish liver [29]. Complement factor H exhibited the highest
upregulation among complement-related factors (14.5-fold), which may inactivate C3b in
the alternative complement pathway [59], suggesting that the host fish were attempting to
modulate the complement response. Two lesser known immune transcripts, lysosomalassociated membrane protein 3 (LAMP-3) and galectin-9, are induced in both blue catfish
following ESC infection [29], and zebrafish following infection with Mycobacterium
marinum [61]. A catfish transcript with highest similarity to lymphocyte antigen 6
complex, locus E (LY6E) [29], which in chicken is a putative disease resistance gene for
Marek’s disease virus [62] were upregulated.
A number of genes not considered acute phase reactants are upregulated in channel
catfish [35], carp, trout and zebrafish include microfibrillar-associated protein 4 [41], Tolllike receptor 5 [33,56], neurotoxin/differentially regulated trout protein [32,33,42,55]
SEC31/high affinity copper uptake protein [32], and SEC61 [42].
A major difference between ESC mediated gene expression in catfish [29,35,63] is the
absence of the iron regulatory hormone hepcidin [64] from the transcriptomic profile of
catfish liver while it is highly upregulated in other teleost livers [32,33,55,56,42]. This
could be useful for future transgenic application as hepicidin is involved in the pathway
that leads to drastically decreased plasma iron levels during infection, a potential host
defense mechanism to deny bacteria access to the critical metal [65]. When plasma iron
levels decrease, it is believed that a feedback mechanism downregulates hepcidin
production in the liver [66,67].
6. Environmental risks and fitness traits
Hypothetically, commercialization of transgenic aquatic organisms on a large scale
could have a variety of ecological implications [68,69] if these fish escape into the natural
environment. Eventual escape of transgenic aquatic organisms is inevitable from a
commercial facility, and the range of receiving ecosystems and possible impacts were
originally thought to be broad.
How can risk of transgenic fish be predicted? Risk of transgenic fish should be no higher
than the risk of domestic fish, and most data indicate that wild fish are more competitive
than domestic fish [70], resulting in the elimination of the domestic fish and their potential
positive or negative impacts. Data from AFLP analysis [71] indicated that domestic
populations of channel catfish in Alabama, USA, have had no genetic impact on wild
populations. However, recent salmonid research indicates that there are situations where
domestic fish can have genetic impact on wild populations. When repeated large-scale
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151
escapes of domestic fish occur, genetic impact can occur just from the sheer force of
numbers. Transgenic fish would make an impact in this scenario, but again the
consequences should not vary much from that of fish genetically altered by traditional
methods. Theoretically, these effects could be positive, negative or neutral. Man impacts
his environment constantly. Different segments of society have different values, and
therefore, the evaluation of the value of impacts can be complex. The assumption is that if
transgenic fish were to establish viable populations in the wild that negative ecological
impacts could occur. Risk is a combination of the probability of an event occurring as well
as the severity of the consequences once an event has occurred [72].
The first aspect of risk concerning transgenic fish is the probability of their survival and
ability to establish themselves if an escape were to occur and that probability revolves
around their fitness. The reproductive performance, foraging ability, swimming ability,
survival and predator avoidance are the key factors determining fitness of transgenic fish,
and should be a standard measurement prior to commercial application to accomplish the
first step of risk analysis. Most available data indicates that transgenic fish are less fit than
non-transgenic fish, and would likely have little if any environmental impact. Extremely
fast growing salmon and loach have low fitness and die [73–75]. Most research on
transgenic fish has been on GH transgenic fish, and subsequently most environmental risk
research has focused on GH transgenic fish. This is pertinent to our topic of use of
transgenic fish to improve disease resistance since GH transfer has the potential to improve
disease resistance.
Several models have been developed that estimate and indicate genetic risk of
transgenic fish. Muir and Howard [76] evaluated a model termed the Trojan gene effect. In
this case, a population can become extinct due to mating preferences for large transgenic
males with reduced fitness placing severe genetic load on the population. Their conclusions
were based on experimental results from GH transgenic medaka in aquaria.
Similarly, Hedrick [77] developed a deterministic model in which a transgene had a
male-mating advantage and a general viability disadvantage, analogous to the Trojan gene
effect, to predict the outcomes of this scenario. Hedrick’s results indicated that for 66.7% of
the possible mating combinations and viability parameters when transgenics invade a
natural population, the transgene increases in frequency, and for 50% of the combinations,
the transgene goes to fixation. The increase in the frequency of the transgene reduces the
viability of the natural population, increasing the probability of extinction of the natural
population.
Muir and Howard [78] again conclude that a transgene is able to spread to a wild
population even if the gene markedly reduces a component of fitness based on data from
medaka harboring a regulatory sequence from salmon fused to the coding sequence for
human growth hormone. The juvenile survival of transgenics was reduced in the laboratory,
but growth rate increased, resulting in changes in the development rate and size-dependent
female fecundity. The important factors in the model were the probabilities of the various
genotypes mating, the number of eggs produced by each female genotype, the probability
that the eggs will be fertilized by the sperm of each male genotype (male fertility), the
probability that an embryo will be a specific genotype given its parental genotypes, the
probability that the fry will survive and parental survival. Muir and Howard’s [78]
interpretation was that transgenes would increase in populations despite high juvenile
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viability costs if transgenes also had sufficiently high positive effects on other fitness traits,
in particular reproduction. Sensitivity analyses indicated that transgene effects on age at
sexual maturity should have the greatest impact on transgene allele frequency changes.
Juvenile viability had the second greatest impact.
A defect in the simulation was the fact that the effect of predation in the wild could not
be included in the model, biasing viability estimates [78]. Although these modeling
experiments based on laboratory data on small model species illustrate potential risk of
transgenic fish, additional weaknesses exist for the models. The environment was artificial,
the mating preference for large males does not exist for many fish, sneaky mating was not
accounted for, the models do not account for genotype–environment interactions which are
likely for growth and other traits, and predation is absent as Muir and Howard [78] indicate.
The overall performance of the fish is not accounted for.
In most fish species, body size does not necessarily result in mating advantages.
Rakitin et al. [79] utilized allozymes and minisatellites markers to determine that male
size, condition factor, and total or relative body-weight loss over the season were not
correlated with the estimated proportion of fry sired by each Atlantic cod male. Similar
results have been observed in salmon [80]. However, Atlantic cod male reproductive
success was affected by female size, with males larger (>25% total length) than
females siring a smaller proportion of larvae [79]; large size was reproductively
disadvantageous.
GH transgenesis has had variable effects on fish reproduction. Fast growing transgenic
tilapia have reduced sperm production, and female GH transgenic Nile tilapia had a lower
gonadosomatic index than non-transgenic siblings in both mixed and separate culture
conditions [81]. Transgenic male gonadosomatic index was higher in mixed culture and
lower in separate culture than that of their non-transgenic siblings. Transgenic channel
catfish and common carp have similar reproduction and rate of sexual maturity compared
to controls [15,82,83]. Spawning success of transgenic channel catfish and controls
appeared similar. When the two genotypes were give a choice in a mixed pond, the mating
was at random, and equal among genotypes [84]. Fecundity is not affected by insertion of
rainbow trout GH cDNA in common carp,and precocious sexual development was not
observed in transgenic common carp.
Reproduction is a complex trait in salmon. Mating success is not determined by size
alone but is also affected by color, body shape, courtship, competition, physiology,
migration ability, environmental effects and genotype–environment interactions. GH
salmon attain normal adult body size, and show advanced hatch time and early growth
[18]. GH salmon show early sexual maturity (one year) in the laboratory, but what age of
maturity they would exhibit in the wild is unknown [85]. Transgenic rainbow trout
experienced early maturation at 2 years of age, but in the same season as the controls.
There is no enhanced adult size in the laboratory, cultured salmon have higher
spermatocrits than transgenics and ‘‘wild hatchery fish’’, transgenics had lower
spermatocrits than controls, and milt was equally competitive among transgenic control,
cultured and wild hatchery fish. However, use of transgenic milt resulted in lower hatch;
GH transgenics had lower spawning and courtship behavior, higher fecundity, but
smaller eggs. Overall, reproductive success of GH transgenic salmon appears to be
adversely effected.
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153
GH transgenic fish could be more competitive in seeking feed. Devlin et al. [86]
examined the motivation of F1 coho salmon (250 g) containing a sockeye
metallothionein-B promoter fused to the type 1 growth gene-coding region to compete
for food. The transgenic coho salmon consumed 2.5 times more contested pellets than
the controls, and the transgenic fish consumed 2.9 times more total pellets that the nontransgenic controls, indicating a high feeding motivation/agressiveness of the transgenic
fish throughout the feeding trials. The shortcomings of this evaluation are that this is a
highly artificial environment and a food type that will not be encountered under natural
conditions. This aggressiveness in seeking food is likely a factor for increasing
vulnerability to predation. Similarly, transgenic tilapia outcompeted controls for
artificial food [87], and had a larger appetite than controls. More importantly, wild
tilapia outcompeted domestic tilapia for food even though this was domestic food. An
important factor in all of these experiments is that the fish are competing for artificial,
not natural, food. For instance, foraging ability of transgenic and control catfish is
similar under these conditions of competition and natural food sources, zooplankton,
and as is the case for most genetic improvement programs, genetically engineered fish
need adequate food to express their potential.
The faster growing transgenic fish could have impaired swimming leading to predator
vulnerability, problems in capturing prey, reduced mating ability for some species and
reduction in competitiveness for any trait requiring speed. Selection for swimming ability
may be one of the primary mechanisms limiting the genetic increase in size of fish and
preventing fish from evolving to larger and larger sizes.
For instance, silversides, Menidia menidia, from Nova Scotia ate more food, had more
efficient feed conversion and grew faster than a population from South Carolina [88],
however, the Nova Scotia strain was more vulnerable to predation than South Carolina
strain, and predation increased with growth rate and feeding rate both within and between
strains [89]. Maximizing energy intake and growth rate has fitness costs in the form of
increased vulnerability to predation [80]. Hungry fast growing fish may take dangerous
risks in the pursuit of food in the presence of predators.
Predator avoidance was slightly better for non-transgenic channel catfish fry and
fingerlings when exposed to largemouth bass, Micropterus salmoides, and green sunfish,
Lepomis cyanellus, than transgenic fry [84,90], GH transgenic salmon have reduced
swimming ability [22,91] and lack of fear of natural predators [92]. GH salmon are willing
to take a bigger risk around predators, and are aggressive feeders. However, predator
avoidance data is conflicting [18,93]. Age and genotype–environment interactions are
important in predator avoidance studies with GH transgenic salmon [18]. Fastest and
slowest growing individuals were eaten more frequently in some, but not all experiments
and this might be related to age effects.
Design of environmental risk/ predation studies in important. Results could be affected
by habitat, artificial versus natural food, same-size fish selection to initiate experiments
could alter genetic make-up of the populations and their behavior. Length of the
experiment is important. In some cases, the salmon GH experiments were conducted for
only 2 days. Dunham et al. [94] demonstrated significant genotype –environment
interaction based on length of experiment for interspecific hybrid and intraspecific
crossbred catfish in angling vulnerability experiments.
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On an absolute speed basis, transgenic coho salmon swam no faster at their critical
swimming speed than smaller non-transgenic controls, and much slower than older nontransgenic controls of the same size [91]. Ostenfeld et al. [95] indicates that coho salmon
containing pOnMTGH1 had altered body contour, centroid size, enhanced caudal peduncle
and enhanced abdominal regions compared to controls. The most prominent alterations
were the change in the syncranium and the head of the transgenics was less elliptical. The
overall body shape is less fusiform for the transgenic coho salmon thus, the decrease in
swimming ability may be a result of loss of hydrodynamics and increased drag coefficients
caused by the altered body shape. This change in body shape might also alter leverage or
efficiency of the muscle movements for swimming. The inferior swimming ability of the
transgenic salmon should cause them to have inferior predator avoidance, inferior ability to
capture food, and inferior ability to migrate to reach the sea or return to reproduce in natural
settings.
All transgenic fish evaluated to date have fitness traits that are either the same or weaker
compared to controls, except for the GH medaka that were not fully evaluated. The
increased vulnerability to predators, reduced swimming ability, lack of increased growth
when foraging, and unchanged spawning percentage of these transgenic fish examples
indicate that some transgenic fish may not compete well under natural conditions, or cause
major ecological or environmental damage. Although transgenic fish may be released to
nature by accident, ecological effects should be unlikely because of this apparent reduced
fitness.
The greatest environmental risk that a transgenic fish would pose is when the gene insert
would allow the transgenic genotype to expand its geographic range, essentially becoming
equivalent to an exotic species. About 1% of such releases of exotics result in adverse
environmental consequences [96]. Altering temperature or salinity tolerance would be
analogous to the development of an exotic species since this would allow the expansion of a
species (transgenic) outside its natural range. Such transgenic research and application
should be avoided. Antifreeze protein genes from winter flounder have been introduced
into Atlantic salmon in an attempt to increase their cold tolerance [97]. If this were
successful, environmental impact is likely. Similarly, if tilapia were made more cold
tolerant, a strong possibility exists for detrimental environmental impact as they would
enable invasion of more temperate climates.
Environmental risk of disease enhanced transgenic fish should be a more
straightforward situation. Disease is much less of problem in the natural environment
compared to the aquaculture environment. The major factors causing mortality in the wild
are starvation and predation, although sometimes devastating epizootics can occur. Most
would take the stance that major disease outbreaks in the wild is not a good event. One
could argue that disease resistant fish, if they were to exist in the natural environment,
might actually promote more stable ecosystems.
7. Transgenic sterilization
Data to date indicates that transgenic fish are inferior to non-transgenic controls for
fitness traits needed for successful establishment in the natural environment. Likely, the
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155
most desirable transgenic genotypes for aquaculture application will be strongly selected
against in natural settings. The greater the phenotypic change for target traits, the greater
the pleiotropic effects on other traits including fitness traits, and the decreased probability
of genetic impact on wild populations. In reality, transgenic fish may be a more acceptable
aquaculture genotype than traditional domestic fish, as high performance transgenics may
actually be eliminated in the natural environment more rapidly than other types of domestic
fish reducing impact on native populations.
However, it will be difficult to prove this hypothesis without an actual escape event,
which is undesirable. Even with strong data indicating the likelihood of low or negligible
environmental risk, virtually all countries will be reluctant to allow commercialization of
transgenic fish because of public perception, the voice of the media and environmental
groups. Therefore, near absolute confinement will be necessary for approval for
commercialization, but most confinement options, physical, chemical and biological are
not 100% fail safe. A potential solution is development of genetic sterilization. Successful
genetic sterilization eliminates almost all environmental issues concerning application of
transgenic fish.
Polyploidy has been proposed as one method to accomplish genetic sterilization,
however, this approach has several shortcomings. Triploid induction is not commercially
feasible for many species, not always 100% effective, requires fertile, diploid brood stock
and triploidy has adverse effects on some economic traits, partially negating some of the
improved performance of the transgenic genotype. Transgenic triploid salmon [17], mud
loach [98] and tilapia [28] have substantial reduction in growth compared to diploid
transgenics, although the triploid transgenic growth is still much better than that of the
controls. Transgenic loach triploids had slightly lower early survival than diploids [98].
Redundant mechanisms could be another option for genetic sterilization. Nam et al. [98]
sterilized transgenic loach with combinations of both triploidy and hybridization, which
had adverse effects on growth. Transgenic diploid loach was 30 times bigger than normal
diploids, hybrid diploids and triploid hybrids. However, transgenic interspecific hybrids
and triploid interspecific hybrids were only 14 larger than the same three non-transgenic
controls.
Transgenic sterilization has the potential to sterilize transgenic fish without the
drawbacks of polypoidy. Transgenic sterilization would almost completely eliminate
environmental risk and may be the most important key for commercialization of transgenic
fish. Still some will argue that the potential would exist for escaped transgenically sterile
fish to disrupt mating of wild conspecifics, thus potentially reducing population numbers,
but this can be evaluated. Unless repeated large-scale escapement occurs, this potential
effect would be temporary. Perfect confinement is not possible for all applications of
transgenic fish. However, the combination of drastically reduced fitness of domestic
transgenic fish, genetic sterilization, transfer of appropriate gene constructs and
appropriate physical confinement will reduce risk to such negligible levels that the
benefits should be much greater than the risks.
Preliminary research on transgenic sterilization has been promising, but this technology is
yet proven 100% effective. Carp beta actin-tilapia salmon type GnRH antisense construct
was injected into Nile tilapia (Norman Maclean, personal communication, [28]. Transgenic
females were crossed with wild type males. A reduction in fertility of about half that of
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non-transgenic control females was observed. Fertility was much more greatly reduced in
transgenic males crossed to control females. In some cases, 0% fertility was obtained with an
average about an 80% reduction in fertility. Once again, traditional selection coupled with
transgenesis may be the answer to obtain the proper transgene performance. Limited data on
transgenic females mated with transgenic males indicated near zero fertility.
Tilapia beta actin-tilapia seabream GnRH antisense construct was transferred to Nile
tilapia, but no reduction in fertility of heterozygous transgenic males and females was
observed (Norman Maclean, personal communication) [28]. Limited data on transgenic
females mated with transgenic males indicated no reduction in fertility. Reciprocal crosses
between seabream and salmon GnRH antisense transgenics gave hatch rates that appeared
to be dictated by the salmon GnRH antisense parent.
Transgenic rainbow trout containing salmon type antisense GnRH from Atlantic
salmon, S. salar, driven by either the salmon GnRH or histone 3 promoter had reduced
levels of GnRH and appear to be sterile [99,100]. Preliminary data indicated that
spermiation of transgenic males only occurred after prolonged treatment with salmon
pituitary extract, whereas normal control males spermiated naturally. Data is still needed
for the fertility of transgenic females to determine if this technology is truly effective.
Another strategy, introduction of ‘‘Sterile Feral, SF,’’ constructs, shows promise and
acts by disrupting embryonic development, thus effectively sterilizing brood stock [101].
Deformities and mortalities were produced with several of the constructs. Gene expression
was reversibly repressed with utilization of doxycycline, dox, as this is an adaptation of the
Tet-OffTM system.
Pilot studies were conducted for this approach utilizing zebrafish and oysters [101].
Actual production of transgenic individuals is not needed for proof of principle, since the
massive amount of SF DNA microinjected into the early embryo simulates the embryo
expressing the constructs themselves. Zebrafish were injected with pzBMP2–As-EGFP
antisense or pBIT(smad)–BMP2ds driven by the smad promoter. For oysters, the promoter
utilized was drosophila heat shock protein coupled with the Hox double stranded genes
double stranded (DS) RNA or dsRNA–zfBMP constructs. In most experiments, 30–60% of
the embryos died or had severe deformitieis, but with the addition of dox there was no
expression of GFP and mortality was reduced to 5% demonstrating the potential of the TetOffTM system. In all of these experiments, controls had 0–5% deformities.
One hundred percent deformities and mortalities were not achieved. This was not
surprising as not all embryos would have received the microinjected genes, and those that
did would all be mosaics of varying degrees and have variable numbers of transgenic cells
in the P1 generation. The reporter genes could also complicate expression, and this has not
been examined.
Templeton [102] evaluated some of these same zebrafish constructs, SF3 (zSMad5
promoter/Bmp2 promoter/dsBmp2 gene and SF4 (zSMad5 promoter/Bmp2 promoter/
zBmp2 gene), in channel catfish to attempt embryonic disruption. Similar to the zebrafish
results, electroporation of these constructs killed channel catfish embryos, and a substantial
percentage of embryos were rescued with the administration of doxycycline. Potential
exists to transgenically sterilize fish and shellfish containing transgenes imparting
enhanced disease resistance, which will help lead to eventual environmentally friendly
application of transgenic aquatic organisms.
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157
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