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PERSPECTIVE / PERSPECTIVE
Genetically modified plants as fish feed
ingredients
Nini Hedberg Sissener, Monica Sanden, A
˚shild Krogdahl, Anne-Marie Bakke,
Lene Elisabeth Johannessen, and Gro-Ingunn Hemre
Abstract: Genetically modified (GM) plants were first grown commercially more than 20 years ago, but their use is still
controversial in some parts of the world. Many GM plant varieties are produced in large quantities globally and are ap-
proved for use in fish feeds both in Norway and the European Union. European consumers, however, are skeptical to fish
produced by means of GM feed ingredients. Concerns have been raised regarding the safety of GM plants, including po-
tential toxicity and (or) allergenicity of the novel protein, potential unintended effects, and risk of horizontal gene transfer
to other species. This review will present the current state of knowledge regarding GM plants as fish feed ingredients, fo-
cusing on fish performance and health as well as the fate of the GM DNA fragments in the fish, identifying limitations of
the current work and areas where further research is needed.
Re
´sume
´:Les plantes ge
´ne
´tiquement modifie
´es (GM) ont commence
´a
`e
ˆtre cultive
´es commercialement il y a plus de
20 ans, mais leur utilisation reste controverse
´e dans certaines re
´gions du globe. Plusieurs varie
´te
´s de plantes GM sont pro-
duites en grande quantite
´a
`l’e
´chelle mondiale et sont accepte
´es dans la pre
´paration de la nourriture de poissons, tant en
Norve
`ge que dans l’Union europe
´enne. Les consommateurs europe
´ens maintiennent cependant une certaine inquie
´tude
concernant les poissons produits par l’usage d’ingre
´dients alimentaires GM. On a manifeste
´des pre
´occupations en ce qui a
trait a
`l’innocuite
´des plantes GM, en particulier a
`la toxicite
´et (ou) l’allerge
´nicite
´potentielles des prote
´ines nouvelles,
aux effets potentiels impre
´vus et au risque de transferts horizontaux des ge
`nes aux autres espe
`ces. Notre re
´trospective re
´-
sume l’e
´tat actuel des connaissances sur les plantes GM utilise
´es comme ingre
´dients dans l’alimentation des poissons,
avec une emphase particulie
`re sur la performance et la sante
´des poissons, ainsi que sur le sort des fragments d’ADN GM
chez les poissons, tout en soulignant les limites des travaux actuels et en identifiant les domaines de recherche addition-
nelle ne
´cessaire.
[Traduit par la Re
´daction]
Introduction
Genetically modified (GM) plants are plants ‘‘whose ge-
netic material is modified in a way which is not found in
nature under natural conditions of crossbreed or natural re-
combination’’ (Gene Technology Act 1993). Genetic modifi-
cation has been used successfully to introduce new
properties in crop plants. Irrespective of the technology
used, integration of the novel DNA happens through so-
called illegitimate recombination (nonhomologous end-joining)
rather than homologous, or site-directed, recombination
(Hansen and Wright 1999; Gelvin 2003). Thus, random in-
sertion of the novel gene might disrupt endogenous gene
expression in the modified plant, which could cause unin-
tended effects, such as changes in levels of macronutrients
and (or) micronutrients, antinutritional factors, or inherent
plant toxicants (Cellini et al. 2004; Somers and Makare-
vitch 2004). If such changes have occurred in the GM
plant, they may affect the value of the plant as a feed in-
gredient for fish. Another concern is that the presence of
the novel GM protein can affect animals consuming the
plant, as exemplified by immunological reactions induced
Received 4 March 2010. Accepted 30 November 2010. Published on the NRC Research Press Web site at cjfas.nrc.ca on 23 February
2011.
J21697
Paper handled by Associate Editor Deborah MacLatchy.
N.H. Sissener,1M. Sanden, and G.-I. Hemre. National Institute of Seafood and Nutrition Research (NIFES), P.O. Box 2029 Nordnes,
5817 Bergen, Norway.
A
˚. Krogdahl and A.-M. Bakke. Norwegian School of Veterinary Science, Department of Basic Sciences and Aquatic Medicine,
Aquaculture Protein Centre, N-0033 Oslo, Norway.
L.E. Johannessen. National Veterinary Institute, P.O. Box 750 Sentrum, N-0106 Oslo, Norway.
1Corresponding author (e-mail: nsi@nifes.no).
563
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in mice (Prescott et al. 2005). According to Kuiper et al.
(2001), a comprehensive safety assessment includes toxic-
ity and allergenicity testing of the novel protein, molecular
characterization of the insert, evaluation of unintended ef-
fects, and potential for horizontal gene transfer to other
species. Safety testing of whole foods is problematic, as
high doses cannot be given to experimental animals with-
out introducing nutritional imbalances, and thus safety mar-
gins for intake are difficult to establish (Kuiper and Kleter
2003).
Since 1996, the proportion of GM crops relative to total
harvest has increased rapidly. In 2008, the percentages of
the global acreage of the crop plants soybean, cotton, maize,
and canola planted with GM varieties were 77%, 49%, 26%,
and 21%, respectively (James 2009). Consequently, it is be-
coming increasingly difficult for fish feed producers to ob-
tain nonmodified varieties of certain plant products
(Kaushik and Hemre 2008; Anonymous 2009).
GM plants in fish feeds
A limited number of fish feeding trials to assess GM
plants as feed ingredients have been conducted (summarized
in Table 1). The studies vary in their focus, as well as fish
species, inclusion level and type of GM ingredients, and du-
ration of the trials. Some studies have utilized the maternal
near-isogenic line of the GM plants in their control diet,
while others have used an unrelated, conventional variety.
In many studies, a fishmeal reference diet has also been
used to establish the normal levels of the measured parameters.
Roundup Ready (RR) soybean
Herbicide tolerance is the most common trait that GM
plants have been modified for, including soybean, maize,
cotton, canola, rice, sugar beet, alfalfa, wheat, cotton, and lin-
seed (GM crop database: www.cera-gmc.org). The glyphosate-
tolerant soybean variety Roundup Ready (RR) is the domi-
nant soy variety on the market (James 2009), which makes
it very relevant for inclusion in animal feeds. This soy ex-
presses the CP4 EPSPS protein, for which no toxic effects
were detected (using protein produced in an E. coli system)
in mice fed high doses, and the protein was found to de-
grade rapidly in simulated gastric fluid and not be homolo-
gous to known allergens or toxins (Harrison et al. 1996). RR
soy has been concluded to be compositionally equivalent to
its nontransgenic parental line, irrespective of herbicide use
and growing location (Padgette et al. 1996; Taylor et al.
1999; McCann et al. 2005).
In the first fish feeding trial conducted with RR soy, cat-
fish (Ictalurus punctatus) fingerlings were fed a diet con-
taining 45% GM soybean or the near-isogenic maternal line
for 10 weeks (Hammond et al. 1996). Fish growth, survival,
feed conversion, and fillet composition exhibited no differ-
ences between the diet groups. Similarly, no differences
were observed in growth, feed performance, and whole
body composition of rainbow trout (Oncorhynchus mykiss)
fed RR soy for 3 months at 15% and 30% inclusion in the
diet (Chainark et al. 2006). No further health or performance
parameters were measured.
Several feeding trials with RR soy have been conducted
with Atlantic salmon (Salmo salar). A challenge in these
studies is that salmon do not tolerate high levels of most
soybean products, depending on the degree of refinement.
They develop an inflammatory response in the distal intestine,
so-called soybean meal-induced enteritis (Van den Ingh et
al. 1991; Baeverfjord and Krogdahl 1996) that can compli-
cate evaluation of GM soybean varieties. Nonetheless, this
has been done, mostly at moderate inclusion levels.
A 3-month study was conducted with Atlantic salmon fed
RR soy at 17% inclusion and a diet with conventional soy
and a fishmeal diet as controls (Hemre et al. 2005; Bakke-
McKellep et al. 2007). The relative size of the spleen was in-
creased in the GM-fed group compared with the non-GM-fed
group, but neither were statistically different from the fish-
meal control group (Hemre et al. 2005). There were no
differences in growth, nutrient utilization, or in other organ
indices. Haematological parameters, plasma nutrient con-
centrations, and leakage of organ-specific enzymes to the
plasma compartment showed similar values in all dietary
groups (Hemre et al. 2005). There was a tendency towards
increased lysozyme activity in the head kidney of the GM-
fed fish (p= 0.06), but no differences in soy-induced in-
flammations or levels of major histocompatibility complex
were detected in the distal intestine of the fish fed the two
soy varieties (Bakke-McKellep et al. 2007). Furthermore,
there were no differences in lysozyme or immunoglobulin
levels in other tissues, nor in EROD (ethoxyresorufin-O-
deethylation) activity in liver (Bakke-McKellep et al.
2007). Samples from the distal intestine of these fish were
used for construction of a suppression subtractive hybrid-
ization (SSH) cDNA library from which clones were se-
quenced and some followed up by qPCR to investigate
differential expression between the diet groups. Only minor
differences were detected (Frøystad et al. 2008). As the
maternal line was not used as control, differences in the
soy cultivars and growing conditions were suggested as po-
tential confounding factors.
The spleen was also substantially larger in GM- versus
non-GM-fed fish in a 28-day trial with 15% and 30% inclu-
sion of RR soy and with the maternal, nonmodified soy-line
used in the control diets (Sagstad et al. 2008). Another dif-
ference observed in this study was lower plasma triacylgly-
cerol (TAG) levels in the GM-fed fish, which may be
explained by a slight difference in saponin levels between
the two soy qualities.
In a study conducted on juvenile Atlantic salmon from
first feeding and with a duration of 8 months, 12.5% hybrid
RR soy was added to the experimental diet, and a commer-
cial soy diet and a fishmeal diet were used as controls
(Sanden et al. 2006). Plasma TAG was found to be lower in
the GM-soy-fed fish compared with those fed conventional
soy. The relative masses of the intestine, spleen, liver, heart,
and brain did not differ between the GM- and non-GM-soy-fed
groups. Investigating the intestinal tract of these fish, San-
den et al. (2005) reported higher cell proliferation in fish
fed the non-GM soy diet compared with GM soy, while
both of these groups were elevated compared with the fish-
meal reference diet, reflecting changes caused by the soy-
induced inflammatory response. No differences were
observed in lysozyme levels or immunoglobulin M in the
intestinal tract. Bakke-McKellep et al. (2008) reported
digestive enzyme activities, active glucose uptake, and
564 Can. J. Fish. Aquat. Sci. Vol. 68, 2011
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SGLT1 (sodium–glucose-linked transporter) protein levels
in the various intestinal regions and found the highest rate
of glucose transport and SGLT1 protein levels in the py-
loric caeca of fish fed GM soy, intermediate levels in
non-GM soy groups, and lowest levels with the fishmeal
groups. Maltase activity in the pyloric caeca was substan-
tially lower in GM-soy-fed than non-GM-soy-fed fish.
Earlier observations of increased spleen size (Hemre et al.
2005; Sagstad et al. 2008) and lowered plasma TAG
(Sanden et al. 2006; Sagstad et al. 2008) were not confirmed
in a 7-month feeding trial with a relatively high inclusion
level (25%) of RR soy and the near-isogenic maternal line
(control), starting in the parr stage and going through the
parr–smolt transformation (Sissener et al. 2009a). In this
study, spleen mass was similar between the groups through-
out the trial, and plasma TAG was found to be higher in the
GM group at all samplings, although the magnitude of this
difference was larger around the time of seawater transfer
than later on in the trial. Furthermore, the relative mass of
the midintestine was found to be lower in the GM group,
something that had not been observed in the previously pub-
lished trials. Other organ indices and plasma enzymes and
nutrients, as well as growth, body composition, haematolog-
ical values, spleen and head kidney lysozyme levels, adapta-
tion to seawater, and stress response were not found to be
different between the diet groups. Histological evaluations
of internal organs revealed decreased liver glycogen deposits
and indications of a more pronounced inflammation status in
the distal intestine of fish fed the GM diet (Sissener et al.
2009b). However, intestinal inflammation was observed in
both diet groups at all sampling points, as would be ex-
pected at the current inclusion level of soybean meal. Fur-
ther analyses are currently being conducted to describe the
differences in the degree of severity of the inflammation
caused by the two soy varieties. When liver protein expres-
sion was compared between the diet groups by means of
proteomics, no apparent biologically meaningful differences
were detected, and the two diet groups were indistinguish-
able by principal component analysis (Sissener et al. 2010a).
A 3-week feeding trial with zebrafish (Danio rerio) fed
Table 1. Overview of fish feeding trials published in the scientific literature with genetically modified (GM) plant products.
GM plant Fish species GM (%) Duration Main effects* Reference(s)
RRS Catfish
(Ictalurus punctatus)
45 10 weeks No differences Hammond et al. 1996
RRS Atlantic salmon
(Salmo salar)
17 3 months Spleen size :;
Lysozyme :
Hemre et al. 2005;
Bakke-McKellep et al. 2007;
Frøystad et al. 2008
RRS Atlantic salmon ~6 8 months Plasma TAG ;;
Cell proliferation ;;
Glucose uptake :
Sanden et al. 2005;
Sanden et al. 2006;
Bakke-McKellep et al. 2008
RRS Rainbow trout
(Oncorhynchus mykiss)
15, 30 3 months No differences Chainark et al. 2006
RRS Atlantic salmon 15, 30 28 days Spleen size :;
Plasma TAG ;
Sagstad et al. 2008
RRS Atlantic salmon 25 7 months Midintestine mass ;;
Plasma TAG :;
Intestinal inflammation :;
Liver glycogen ;
Sissener et al. 2009a;
Sissener et al. 2009b;
Sissener et al. 2010a
RRS Zebrafish (Danio rerio) 25 3 weeks Liver RNA yield ;Sissener et al. 2010b
MON810 Atlantic salmon ~6 8 months No differences Sanden et al. 2005;
Sanden et al. 2006;
Bakke-McKellep et al. 2008
MON810 Atlantic salmon 15, 30 82 days Feed intake ;;
Growth ;;
Liver size :;
Distal intestine mass :;
Glucose uptake :;
SOD :;
CAT ;;
Granulocytes :
Hemre et al. 2007;
Sagstad et al. 2007;
Frøystad-Saugen et al. 2009
MON810 Zebrafish 20 3 weeks Growth :;
SOD ;
Sissener et al. 2010b
GM cotton Catfish 20 8 weeks Fillet protein ;Li et al. 2008
RR canola Rainbow trout 5, 10, 15,
20
NA{Protein retention :Brown et al. 2003
GM lupin Red seabream
(Pagrus auratus)
60 40 days Growth :Glencross et al. 2003
Note: Only studies evaluating the effects of GM plants as feed ingredients are included (not studies focusing on the traceability of DNA).
RRS, Roundup Ready soy; TAG, triacylglycerol; SOD, superoxide dismutase; CAT, catalase.
*Differences between fish fed GM to fish fed non-GM plants: (:) indicates that the parameter in question was higher in the GM-fed fish, while
(;) indicates lower values in the GM-fed group. Further details on the changes and confounding factors in the studies are discussed in the text.
{Duration of study is not clear from the paper.
Sissener et al. 565
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RR soy (25% of diet) and the maternal near-isogenic line
has also been published (Sissener et al. 2010b). Liver RNA
yield tended to be higher in fish fed non-GM compared with
GM soy (p= 0.06), and there was a significant interaction
effect between soy variety and the sex of the fish (p=
0.003). The females in the non-GM soy group seemed to
have larger livers than the females in the GM soy group,
while this was not the case for the males. There was also an
interaction effect between soy variety and sex on liver
superoxide dismutase (SOD)-1, with lowered transcription
levels in females but not in males fed GM soy. These inter-
actions indicate a difference in how the two soy varieties af-
fect the male and female zebrafish, possibly by differentially
affecting sex hormones. Soy is known to contain phytoestro-
gens (soy isoflavones) that can affect reproduction and vitel-
logenin levels in fish (Pelissero et al. 1991; Kaushik et al.
1995; Kelly and Green 2006). Unfortunately, these com-
pounds were not measured in the soy used for this trial.
There were no differences in mass, length, or condition fac-
tor, nor in the mRNA transcription of a range of other
stress-related genes, such as heat shock protein 70 (hsp70),
hsp90, DNA-repair protein, and glutathione peroxidase.
The lack of reproducibility or even contradictory results
observed between the different fish feeding trials summar-
ized above, even between trials using the same fish species
(with salmon being the most extensively studied), suggest
that these effects are not caused by the GM protein. How-
ever, they may be related to unintended effects of the ge-
netic modification, which can result in variations in levels
of antinutritional factors, antigens, metabolites, or other un-
identified factors in the plants. Variations in such compo-
nents also occur between batches of conventionally
produced plants, as evidenced by varying levels of soybean
meal-induced intestinal inflammation in salmon fed soy
grown in different locations around the world (Ura
´n et al.
2009). None of the differences observed between the diet
groups have been identified as adverse, as they appear to be
within the normal physiological ranges for the species, at
least as far are these ranges have been identified in these
fish species. A large range of health- and performance-related
parameters have been investigated in fish, mainly salmon,
fed RR soy, and the overall conclusion is that only minor,
rather variable differences are seen, and none of the studies
showed differences in growth or nutrient retention. Conse-
quently, RR soy appears to be an equally suitable feed in-
gredient as conventional soy at dietary inclusion levels
appropriate for the assessed fish species.
Bacillus thuringiensis (Bt)-maize
In the category of insect-resistant GM plants, plants ex-
pressing different Cry-proteins have been developed (www.
cera-gmc.org). Naturally produced by the spore-forming soil
bacterium Bacillus thuringiensis, these proteins are often re-
ferred to as Bt-proteins or Bt-toxins. The different Cry-proteins
bind to specific receptors in the midintestine of their target
insects, where they cause damage to the intestinal wall by
forming pores and changing the permeability of epithelial
cells, eventually resulting in death (Knowles 1994).
Administered to mice in a dose of 4000 mgkg–1 in the
feed, the Cry1Ab protein caused no signs of acute toxicity
(Sanders et al. 1998), but studies in mice using Cry1Ac
have observed binding to mucosa, hyperpolarization of in-
testinal tissue, and stimulation of antibody production
(Va
´zquez-Padro
´n et al. 1999 and 2000; Moreno-Fierros et
al. 2000).
Regarding compositional equivalence, 10 Bt-corn hybrids
were found to have higher content of lignin, a major struc-
tural component of plant cells, than their near-isogenic lines
(Saxena and Stotzky 2001). Levels of mycotoxins in corn
kernels may cause differences in nutritional value of Bt ver-
sus conventional maize (Munkvold et al. 1997). Despite
large variations between locations and seasons, mycotoxin
levels have generally been observed to be reduced in Bt-
maize, as insect damage can predispose for fungal growth
(e.g., Dowd 2000; Bakan et al. 2002; Papst et al. 2005).
This has been used to explain higher mass gain in the GM-
fed group in some studies with various production animals
fed Bt-maize (reviewed by Flachowsky et al. 2005),
although cause–effect relationships were not established.
Sanden et al. (2006) evaluated two different MON810 hy-
brids along with nonmodified controls in an 8-month feed-
ing trial with Atlantic salmon juveniles from first feeding
onwards. The inclusion level of the different maize types
was 12% (GM content about 6%). Differences in response
parameters in the fish were seen between the different maize
types, but none appeared to be related to whether the maize
was GM or not. The evaluations included assessment of
growth; organ masses; plasma parameters; histological, di-
gestive, metabolic, and immunological parameters, as well
as intestinal cell proliferation (Sanden et al. 2005, 2006;
Bakke-McKellep et al. 2008).
In another study, Atlantic salmon postsmolt fed GM
maize (MON810) at 15% and 30% of the total diet had sub-
stantially reduced feed intake, growth rate, and final mass
compared with fish fed non-GM maize (Hemre et al. 2007).
Furthermore, major differences were revealed in organ indi-
ces: the relative mass of both liver and distal intestine was
increased in the fish fed GM maize. Some differences were
observed in head-kidney and spleen somatic indices as well,
but these were not dose related and not considerably differ-
ent between the GM- and non-GM-fed groups as a whole.
No differences were observed during histological evalua-
tions of the same organs. Maltase activity in the midintesti-
nal segment of fish fed the 30% GM diet was higher than
the 30% non-GM group. Uptake of glucose in pyloric caeca
was also substantially higher in fish fed the GM diet, reflect-
ing findings in salmon fed GM soy (Bakke-McKellep et al.
2008). Fish health in this trial was evaluated focusing on
stress- and immune-response biomarkers (Sagstad et al.
2007). SOD had higher activity in liver and distal intestine,
while catalase (CAT) showed substantially lower liver activ-
ity in fish fed GM maize. HSP70 was significantly higher in
the liver of fish fed GM maize compared with the fishmeal
reference diet, while the non-GM maize group exhibited in-
termediate levels. The differences in activity and protein
levels of CAT, SOD, and HSP70 were not reflected in levels
of mRNA coding for these proteins. Differential count of
white blood cells revealed a considerably higher proportion
of granulocytes in the blood of fish fed GM maize. The au-
thors suggested that the GM maize MON810 caused changes
in the immune response with a related mild cellular stress
response. Altered liver metabolism was also indicated based
566 Can. J. Fish. Aquat. Sci. Vol. 68, 2011
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on the higher liver index and changes of CAT, HSP70, and
SOD. Construction of an SSH-library from distal intestine,
followed up by qPCR, revealed only minor differences be-
tween the diet groups in gene transcription (Frøystad-Saugen
et al. 2009). Microarray data from liver and distal intestine
of these fish are currently being evaluated.
In a 3-week zebrafish feeding trial with 20% inclusion of
GM maize (MON810) and the maternal line as control, us-
ing the same batch of maize as in the salmon trial described
above, better growth was observed in the GM-fed group
(Sissener et al. 2010b). Additionally, reduced SOD-1
mRNA levels and a tendency (nonsignificant) towards re-
duced hsp70 mRNA levels were found in liver of fish fed
GM compared with non-GM maize. The discrepancy of
these results with those obtained in salmon (Hemre et al.
2007; Sagstad et al. 2007) is hard to explain, but could be
due to differences in species, developmental stage, duration
of the trials, maize inclusion levels, or feed processing.
Compared with RR soy, the data on use of Bt-maize in
fish feeds are scarce, and the results in the above mentioned
trials differ widely. Thus, further research is needed to con-
clude on whether or not Bt-maize fed to fish can have dif-
ferent effects on growth, stress, and immune responses
compared with nonmodified maize.
GM cotton
Different varieties of GM cotton have been evaluated in
diets for channel catfish (Li et al. 2008). The four GM cot-
ton varieties evaluated were Roundup Ready Flex (ex-
pressing the CP4 ESPS protein), Bollgard Roundup
Ready (expressing both Cry1Ac and CP4 EPSPS),
Bollgard II Roundup Ready (expressing Cry1Ac,
Cry2Ab2, and CP4 EPSPS), and Bollgard II Roundup
Ready Flex (expressing Cry1Ac, Cry2Ab2, and CP4
EPSPS). The three latter cotton varieties are crosses of two
GM varieties, so-called stacked events combining both her-
bicide tolerance and insect resistance in the same plants. Nu-
tritional evaluations of cotton seed meal (CSM) from these
plants were conducted in three separate feeding experiments,
all of which lasted for 8 weeks and included non-GM CSM
from a genetically similar control as well as several com-
mercial varieties as references. All diets contained 20%
CSM. Chemical analyses of the various CSM varieties re-
vealed differences in proximate composition and gossypol
levels in all three trials, but these were to some extent bal-
anced for in the feed formulation. The parameters examined
were growth, survival, feed efficiency, and fillet composi-
tion. Bollgard RR and Bollegard II RR were both
found to cause lower fish fillet protein content than the ge-
netically similar, nonmodified CSM, while only Bollegard
RR-fed fish were substantially different from fish fed the
commercial varieties as well. Despite this difference, the au-
thors conclude that all the GM CSMs are nutritionally
equivalent to nonmodified CSM (Li et al. 2008).
RR canola
Two lines of RR canola have been tested in diets for rain-
bow trout: modification events GT200 and GT73, both con-
taining the gene for CP4 EPSPS and a glyphosate oxidase
gene from Ochrobactrum anthropi that catalyses the break-
down of glyphosate. These were included in fish diets in a
regression design at 5%–20% and compared with the paren-
tal line as well as a reference diet not containing canola
(Brown et al. 2003). The GT200 line was equivalent to non-
modified canola as judged by fish performance, while the
GT73 line resulted in increased protein retention, increased
body protein and moisture, and reduced body lipid compared
with the parental line. This might be due to small differen-
ces in protein content and nitrogen solubility between the
canola lines, which does not seem to have been balanced
for in the formulation of the diets. Specific information con-
cerning this, as well as compositional analysis of the diets,
was not supplied. The authors conclude that glyphosate-tolerant
canola is equivalent to conventional canola for use in trout
diets (Brown et al. 2003).
Lupin with increased methionine
The first GM plants commercialized were aimed at im-
proving agronomical traits, such as tolerance to herbicides
or resistance to insect attack. More elaborate modifications,
aimed at changing the nutrient profile of the plants, are
often referred to as ‘‘second generation’’ GM plants. The
only second generation GM plant with published results in
fish diets is a GM lupin variety in diets for juvenile red
seabream (Pagrus auratus) (Glencross et al. 2003). This lu-
pin line was modified to express a sunflower seed albumin
gene leading to increased levels of methionine (Molvig et
al. 1997), but has not been commercialized. Two commer-
cial lupin varieties were used as reference, one of which
was very close in proximate composition to the GM variety.
A diet with this latter non-GM lupin variety fortified with
crystalline methionine was also included in the experimental
design. Apart from poorer growth in fish fed one of the non-
GM lupin varieties, which had much lower protein content
than the others, no differences were observed in an initial
experiment with high protein diets and ad libitum feeding
(Glencross et al. 2003). In a subsequent feeding trial with
subsatietal pair-feeding and protein-restrictive diets, a bene-
ficial effect on growth was seen with both the GM diet and
the non-GM diet with added crystalline methionine when
compared with the nonfortified non-GM variety.
GM plants of the future as fish feed ingredients
To our knowledge, no work has been conducted on com-
mercialized second generation GM plants as fish feed ingre-
dients. Attempts to tailor nutrient composition are likely to
give unintended effects, as further changes are made in met-
abolic pathways and nutrient balance (Larkin and Harrigan
2007). This may cause altered levels of undesirable substan-
ces (e.g., anti-nutritional factors) and reduced growth in ex-
perimental animals (Bo
¨hme et al. 2005; Bo
¨hme et al. 2007).
However, these GM plants do not only present challenges
for the animal feed industry, but also opportunities for novel
plant products better suited for specific animal species, such
as fish. Few such GM plants are commercially available, but
many are under development and can be expected to be in-
troduced to the market in the coming years.
Replacement of fish oil and fishmeal by lipid and protein
from vegetable sources is a necessity to make the aquacul-
ture industry sustainable (FAO 2005). However, fish oil re-
placement may affect the product quality and health benefits
for the consumer, as the fish fillet will contain less of the
Sissener et al. 567
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marine long-chained polyunsaturated omega-3 fatty acids.
Plants modified by introducing genes from microalgae to
produce omega-3 fatty acids are under development, and
commercial production is targeted to begin in 2015 (Watkins
2009). Monsanto (http://www.monsanto.com/Pages/default.
aspx) has also been working on production of long-chain
omega-3 fatty acids in canola (Ursin 2003).
Amino acid composition of plant proteins is another way
in which plants can be modified to be more attractive as fish
feed ingredients. Maize with increased level of lysine is al-
ready on the market (LY038) and has been tested with pos-
itive results in broilers (Lucas et al. 2007). Corn gluten is a
promising protein source for use in fish, but low lysine level
is considered a limiting factor (Aslaksen et al. 2007). Refer-
ence diets must be designed to contain lysine levels below
what is required for optimal performance to avoid similar
problems as experienced by Glencross et al. (2003), and an
additional reference diet spiked with crystalline lysine to the
same level as present in the GM plant would be a natural
control. Such modifications could be very useful, as amino
acids from intact protein seem to be utilized better than free
amino acids in fish (Davies and Morris 1997; Sveier et al.
2001). Similarly, the usefulness of other plants proteins
could be increased by genetic modification, such as in-
creased methionine content in legumes (Mu
¨ntz et al. 1998).
Antinutritional factors severely hamper performance espe-
cially in salmonid fish fed soybean meal (which is a pre-
ferred feed ingredient owing to availability and a relatively
good amino acid profile considering the requirements of the
fish). There are also numerous antinutrients in other plant
products limiting their use in fish feeds (Francis et al.
2001). Many of these compounds have protective roles in
plants; for example, for cotton, a variety without gossypol
was developed through traditional breeding, but was not suc-
cessful as it was highly prone to insect attack. With genetic
modification, using antisense and RNAi to silence target
genes in specific tissue, it was possible to eliminate gossy-
pol production only in the seeds of cotton (Sunilkumar et
al. 2006).
Uptake of transgenic DNA
One of the concerns related to the use of GM feed ingre-
dients is the fate of the inserted DNA sequence, consisting
of a transcription promoter, a coding sequence for the gene
of interest, and an expression terminator. This DNA is often
coupled to a marker that is used to identify successfully
transformed cells, for example, antibiotic-resistant genes.
There is concern related to the possible horizontal gene
transfer (HGT) of intact antibiotic-resistant genes to the mi-
croflora of the intestinal tract of animals eating GM ingre-
dients. HGT is defined as the transfer of genetic material
directly to a living cell or an organism followed by its ex-
pression, a common phenomenon among prokaryotes
(van den Eede et al. 2004). Most scientists conclude that
these events occur at such low frequencies in nature, if at
all, so that as long as one uses genes conferring resistance
to antibiotics of limited clinical importance and to which re-
sistant bacteria are already widespread both in the human
gut and the environment (such as the commonly used nptII
marker), there is no risk attached (Gay and Gillespie 2005;
European Food Safety Authority 2009). However, there are
some that disagree and criticize the sensitivity of studies
conducted to detect these events. Several methods have
been developed for alternative marker systems or removal
of marker genes before release of GM plants (Hohn et al.
2001; Cuellar et al. 2006). Of the commercialized GM
plants used in the studies described in this paper, Bollogard
cotton is the only one containing an antibiotic resistance
marker (nptII).
However, concern is also linked to the possibility of HGT
to (eukaryotic) animals. There are numerous articles report-
ing on the fate of DNA from GM plants in mammals (e.g.,
Schubbert et al. 1998; Hohlweg and Doerfler 2001), and
questions have been raised regarding the fate of GM DNA
in edible products such as milk, meat, and eggs (Einspanier
et al. 2001).
Modern salmonid diets produced by extrusion are sub-
jected to mixing, shearing, and heating under high pressure.
Despite this, GM DNA fragments have been detected in ex-
truded fish diets (Sanden et al. 2004; Chainark et al. 2008).
In the gastrointestinal tract (GIT) of animals, dietary DNA
will be broken down to smaller monomers through the ac-
tion of gastric acids and pancreatic and intestinal epithelial
cell nucleases. A number of factors may influence the per-
sistence and stability of DNA in the intestine, including the
feed matrix within which the DNA is contained; for exam-
ple, complex carbohydrates have been found to have a pro-
tective role (Palka-Santini et al. 2003). DNA fragments that
have survived feed processing and the digestive enzymes of
the GIT may potentially be absorbed into the intestinal mu-
cosa and subsequently transported into the rest of the body.
However, mechanisms of absorption are not known. Trans-
cellular absorption of undigested macromolecules, mostly
proteins, has been demonstrated in the fish intestine
(Rombout et al. 1985; Buddington et al. 1997). The paracel-
lular route involving a direct diffusion of molecules between
epithelial cells could be another possible pathway (McLean
and Ash 1987). The paracellular route is often associated to
particular situations, such as intestinal pathologies (Sire and
Vernier 1992). Additionally, the permeability of the intestine
is affected by acute stress (Sundell et al. 2003), dietary
lectins (Gatlin et al. 2007), saponins, and trypsin inhibitors
(McLean et al. 1990). Dietary DNA might also be co-transported
across intestinal cells in a complex with various feed com-
ponents, as found for oligonucleotides conjugated to li-
gands such as cholesterol (Vlassov et al. 1994). In vitro
experiments using the human intestinal cell line CaCo-2
suggests that unconjugated DNA fragments can be trans-
ported across the intestine by a transcellular route
(L.E. Johannessen, K. Berdal, A. Holst-Jensen, unpublished
data). Transport of DNA across CaCo-2 cells is 1000–
10 000 times higher in the apical to basolateral direction
than in the opposite direction, indicating active transport.
Only few studies have investigated the fate of foreign
DNA ingested by fish. In Atlantic salmon force-fed high
copy numbers of polymerase chain reaction (PCR)-amplified
DNA fragments, foreign DNA fragments were detected in
liver, kidney, and blood (Nielsen et al. 2005). Similarly, in-
travenously injected PCR-amplified DNA was detected in
muscle, liver, kidney, and blood of salmon (Nielsen 2006).
By means of in situ hybridization, dietary DNA from GM
soy was identified in the epithelial cells in the salmon intes-
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tine, in the cells’ vacuolar system, and in the lamina propria
in some samples (Sanden et al. 2006). A recent study inves-
tigating the presence of the Cauliflower mosaic virus
(CaMV) 35S promoter from GM soybeans reported DNA
fragments in the leukocytes, head-kidney, and muscle of
rainbow trout, but not 5 days after changing to a non-GM
diet (Chainark et al. 2008). In tilapia (Oreochromis niloti-
cus) fed GM soy diets, fragments of the CaMV 35S pro-
moter were detected in a few muscle samples after
continuous feeding, while none were detected 2 days after
changing to a non-GM diet (Suharman et al. 2009). How-
ever, in another study with tilapia fed GM soy, GM DNA
was still detected in all organs investigated after 2 weeks of
fasting (Ran et al. 2009).
In a zebrafish study, dietary DNA fragments from rubisco
(a multicopy plant gene) and from MON810 were detected
in several organs, while RR soy DNA fragments were not
detected (Sissener et al. 2010b). This is in agreement with
studies in other species, supporting the observation that diet-
ary DNA uptake is not a specific property of GM DNA se-
quences, but occurs also with DNA fragments from
conventional feed ingredients (Hohlweg and Doerfler 2001;
Phipps et al. 2003; Reuter and Aulrich 2003). Furthermore,
the GM insert does not modify the uptake of other dietary
DNA fragments (Reuter and Aulrich 2003; Mazza et al.
2005). However, there seems to be differences in the degra-
dation or uptake of different DNA fragments, possibly de-
pending on nucleotide sequence, methylation patterns, or
simply the feed matrix (Sissener et al. 2010b).
Common for all these studies is that some dietary DNA
seems to survive the harsh conditions of feed preparation
and also that dietary DNA fragments can be taken up in the
intestine and be detected in internal organs of the fish. The
fragments detected have generally been below a size likely
to code for functional proteins (Jonas et al. 2001), but longer
fragments were not searched for. The data currently avail-
able do not assess the potential for horizontal gene transfer,
either to the fish itself or to microflora in the intestine. Ad-
ditioanlly, the mechanisms and the possible function (posi-
tive or negative) of such uptake in fish or other animal
species are not known. Further research on specific mecha-
nisms of dietary DNA uptake specifically aiming to recog-
nize what intestinal cell types are involved is needed.
Feeding trials with fish: limitations and
challenges
The feeding trials discussed in this paper vary widely in
their focus, inclusion of GM ingredients, duration of trials,
choice of reference diets, and what response parameters are
measured in the fish. There is no general consensus regard-
ing these issues, nor on whether feeding trials are indeed
necessary (Knudsen and Poulsen 2007; European Food
Safety Authority 2008). The fact that some fish species, es-
pecially carnivorous fish such as salmon, are very sensitive
to certain plant components compared with terrestrial pro-
duction animals can certainly be used as an argument in fa-
vour of evaluating GM feed ingredients in fish. However,
the general lack of information regarding which plant anti-
nutrients or other plant components (regardless of whether
the plant is GM or not), and at what level, will cause prob-
lems in fish is still a drawback. That some antinutrients in-
teract with each other, affecting tolerance levels and what
detrimental effects may occur, makes it even more difficult
to predict effects in fish (Francis et al. 2001). Thus, it would
be difficult to predict effects solely based on chemical anal-
ysis of the GM plant. Since unintended effects with subse-
quent differences in endogenous components in GM
compared with non-GM plants may account for differences
in responses observed in fish feeding trials, more research is
needed to identify such components and their effects in fish.
Regarding what endpoints to assess in the fish, in a time
with increasing focus on fish welfare, focusing solely on
production parameters such as survival, growth, and fillet
composition might not be enough for sceptical consumers.
More sensitive parameters than growth and survival need to
be included in the evaluation to be able to assess health sta-
tus and detect early stage differences that might become ad-
verse using suboptimal diets for prolonged periods of time
(e.g., throughout the life cycle of the fish in an aquaculture
setting). Untargeted screening techniques such as microarray
and proteomics could be useful approaches both as tools to
screen for unintended effects in the GM plant – GM feed
and in fish target organs.
Another problem is how and what to use as a control in
the trial. The near-isogenic maternal line grown under simi-
lar conditions as the GM crop is recognized as the recom-
mended control (European Food Safety Authority 2006).
Despite this, differences in the proximate composition be-
tween GM and maternal non-GM lines of CSM, canola, and
soy used in fish trials described in this paper were identified
(Brown et al. 2003; Li et al. 2008; Sagstad et al. 2008). This
shows that even such closely related and produced crops are
not identical in composition. The question then arises on
how to interpret minor differences found in measured pa-
rameters in the experimental animals. How can one distin-
guish whether these are due to the GM protein, unintended
effects of the genetic modification, or unrelated random dif-
ferences in plant cultivars that might vary from one growing
season or location to the next? Faced with this dilemma, it
might be preferable to use several commercial non-GM cul-
tivars as controls in the safety assessments of GM ingre-
dients. Thus, one could establish if the parameters measured
in animals fed GM plants are within the normal range of an-
imals fed unmodified crops (European Food Safety Author-
ity 2008). The disadvantage is that this would greatly
increase the cost of trials, as well as the number of experi-
mental animals needed. In addition to establishing whether
a response is within the normal range, there is the issue of
false discoveries. When many different parameters are
tested, there is a greater risk that significant changes are de-
tected because of chance alone. (On the other hand, real ef-
fects might not be detected because of insufficient power of
the experiment.) Wilson et al. (2001) provide a list of ques-
tions for assessing the biological relevance of differences
detected in toxicological studies, which could also be useful
for GM safety assessment: (i) whether the trends are dose-
related, (ii) whether they are reproducible, (iii) whether there
is a relationship to other findings, and (iv) whether the mag-
nitude of the differences suggest that they are biologically
important.
Regarding diet formulation, it is crucial to ensure that the
Sissener et al. 569
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diets are equivalent both in proximate composition and in
other compounds that are relevant to the growth and health
of the experimental animals. However, there will always be
compounds in the feed that are not analysed, and there will
be differences in the dietary ingredients that are not possible
to balance in the feed formulation. Examples include antinu-
trients such as phytoestrogens or saponins, variable levels of
naturally occurring compounds such as mycotoxins, or her-
bicide and pesticide residues. Residues of these compounds
might explain some of the differences observed between
feeding trials, or at least knowing whether or not there are
such differences would better enable us to separate ‘‘GM ef-
fects’’ from effects caused by confounding factors. Further-
more, the levels of GM protein expression in the plant
ingredients are rarely quantified, and differences between
different batches of GM plants in this respect might also ex-
plain some of the discrepancies observed between feeding
trials. Quantifying the transgenic protein and using an addi-
tional control diet spiked with the transgenic protein would
be necessary to be able to distinguish between unintended
and intended effects of the genetic modification (European
Food Safety Authority 2008).
Adequate control diets already present a challenge in GM
safety assessments, but this problem will most likely in-
crease in the future, with stacked event GM plants (crosses
of two or several modification events) and second generation
GM plants with intended changes in nutrient composition.
Conclusions
Among the herbicide-tolerant GM plants, only RR plants
have been evaluated for use in fish feeds, most extensively
in salmon, but some data also exist from channel catfish,
rainbow trout, and zebrafish. RR soy is the most extensively
evaluated and appears to possess similar qualities as nonmo-
dified, conventional soy products as a feed ingredient for
fish. Minor or no differences were observed in the different
trials, and these were not consistent from one trial to the
next. Results from RR canola and cotton also show no or
minor differences between diet groups.
Among the insect-resistant (Bt) plants, data are more scant
and inconclusive, both as less research has been carried out
and more pronounced differences have been observed be-
tween fish fed Bt-maize and conventional maize. The cause
of these differences is not clear and should be followed up,
including a thorough evaluation of possible effects of the
Bt-protein on the fish intestine and on growth, cellular stress,
and immune responses. Bt-CSM showed minimal differences
from conventional CSM on growth, survival, feed efficiency,
and fillet composition in channel catfish, but more specific
health parameters were not evaluated.
The difference in results from the feeding trials conducted
with RR soy and Bt-maize clearly shows that evaluation of
one GM variety cannot automatically be extrapolated to
make conclusions on the safety of other GM plants. Thus,
each modification event should be considered unique, with
the insertion of target genes coding for proteins that have
different characteristics and with the potential occurrence of
different unintended changes in nutrient or antinutrient com-
position. Case-by-case assessment has been advocated by
others (Kuiper et al. 2001; Larkin and Harrigan 2007) and
is the norm both in Norway (Norwegian Food Safety
Authority) and the European Union (European Food Safety
Authority).
Second generation GM plants have potential, since plant
products with a nutritional profile more tailored to the spe-
cific needs of the fish can be developed, but they will also
present additional challenges regarding safety assessment.
Dietary DNA fragments, including transgenic ones, with-
stand fish feed processing and intestinal digestion and can
be absorbed by the intestine and further distributed to vari-
ous tissues in the fish. The present results give no reason to
suspect that transgenic sequences are taken up more fre-
quently than regular plant DNA, or that this uptake causes
any negative effects for the fish. However, the mechanisms
and possible function of dietary DNA uptake in the intestine
are unknown, as are types of cells involved and potential
consequences of this uptake. The possibility for horizontal
or vertical gene transfer has not been evaluated in fish.
Acknowledgments
The work on GM ingredients in fish feeds conducted by
the authors of this paper have been financed mainly by the
Research Council of Norway through two consecutive proj-
ects (grants Nos. 142474 and 172151) from 2001 to 2009.
References
Anonymous. 2009. Gennytt, 5. Available from http://www.oikos.
no/newsread/news.asp?docid=11097 [accessed 15 December
2009].
Aslaksen, M.A., Kraugerud, O.F., Penn, M., Svihus, B.,
Denstadli, V., Jørgensen, H.Y., Hillestad, M., Krogdahl, A
˚., and
Storebakken, T. 2007. Screening of nutrient digestibilities and
intestinal pathologies in Atlantic salmon, Salmo salar, fed diets
with legumes, oilseeds, or cereals. Aquaculture, 272(1–4): 541–
555. doi:10.1016/j.aquaculture.2007.07.222.
Baeverfjord, G., and Krogdahl, A
˚. 1996. Development and regres-
sion of soybean meal induced enteritis in Atlantic salmon,
Salmo salar L., distal intestine: a comparison with the intestines
of fasted fish. J. Fish Dis. 19(5): 375–387. doi:10.1111/j.1365-
2761.1996.tb00376.x.
Bakan, B., Melcion, D., Richard-Molard, D., and Cahagnier, B.
2002. Fungal growth and fusarium mycotoxin content in iso-
genic traditional maize and genetically modified maize grown
in France and Spain. J. Agric. Food Chem. 50(4): 728–731.
doi:10.1021/jf0108258.
Bakke-McKellep, A.M., Koppang, E.O., Gunnes, G., Sanden, M.,
Hemre, G.I., Landsverk, T., and Krogdahl, A
˚. 2007. Histologi-
cal, digestive, metabolic, hormonal and some immune factor re-
sponses in Atlantic salmon, Salmo salar L., fed genetically
modified soybeans. J. Fish Dis. 30(2): 65–79. doi:10.1111/j.
1365-2761.2007.00782.x.
Bakke-McKellep, A.M., Sanden, M., Danieli, A., Acierno, R.,
Hemre, G.I., Maffia, M., and Krogdahl, A
˚. 2008. Atlantic sal-
mon (Salmo salar L.) parr fed genetically modified soybeans
and maize: histological, digestive, metabolic, and immunological
investigations. Res. Vet. Sci. 84(3): 395–408. doi:10.1016/j.rvsc.
2007.06.008.
Bo
¨hme, H., Hommel, B., and Flachowsky, G. 2005. Nutritional as-
sessment of silage from transgenic inulin synthesizing potatoen
for pigs. J. Anim. Feed Sci. 14: 333–336.
Bo
¨hme, H., Rudloff, E., Scho
¨ne, F., Schumann, W., Hu
¨ther, L., and
Flachowsky, G. 2007. Nutritional assessment of genetically
570 Can. J. Fish. Aquat. Sci. Vol. 68, 2011
Published by NRC Research Press
Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by Institute of Marine Research (Norway) on 09/03/12
For personal use only.
modified rapeseed synthesizing high amounts of mid-chain fatty
acids including production responses of growing-finishing pigs.
Arch. Anim. Nutr. 61(4): 308–316. doi:10.1080/
17450390701432704.
Brown, P.B., Wilson, K.A., Jonker, Y., and Nickson, T.E. 2003.
Glyphosate tolerant canola meal is equivalent to the parental
line in diets fed to rainbow trout. J. Agric. Food Chem. 51(15):
4268–4272. doi:10.1021/jf034018f.
Buddington, R.K., Krogdahl, A
˚., and Bakke-McKellep, A.M. 1997.
The intestines of carnivorous fish: structure and functions and
the relations with diet. Acta Physiol. Scand. 161: 67–80.
Cellini, F., Chesson, A., Colquhoun, I., Constable, A.,
Davies, H.V., Engel, K.H., Gatehouse, A.M.R., Karenlampi, S.,
Kok, E.J., and Leguay, J.J. 2004. Unintended effects and their
detection in genetically modified crops. Food Chem. Toxicol.
42(7): 1089–1125. doi:10.1016/j.fct.2004.02.003.
Chainark, P., Satoh, S., Hino, T., Kiron, V., Hirono, I., and
Aoki, T. 2006. Availability of genetically modified soybean
meal in rainbow trout Oncorhynchus mykiss diets. Fish. Sci.
72(5): 1072–1078. doi:10.1111/j.1444-2906.2006.01258.x.
Chainark, P., Satoh, S., Hirono, I., Aoki, T., and Endo, M. 2008.
Availability of genetically modified feed ingredient: investiga-
tions of ingested foreign DNA in rainbow trout Oncorhynchus
mykiss. Fish. Sci. 74(2): 380–390. doi:10.1111/j.1444-2906.
2008.01535.x.
Cuellar, W., Gaudin, A., Solo
´rzano, D., Casas, A., N
˜opo, L.,
Chudalayandi, P., Medrano, G., Kreuze, J., and Ghislain, M.
2006. Self-excision of the antibiotic resistance gene nptII using
a heat inducible Cre-loxP system from transgenic potato. Plant
Mol. Biol. 62(1–2): 71–82. doi:10.1007/s11103-006-9004-3.
Davies, S.J., and Morris, P.C. 1997. Influence of multiple amino
acid supplementation on the performance of rainbow trout, On-
corhynchus mykiss (Walbaum), fed soya based diets. Aquacult.
Res. 28(1): 65–74. doi:10.1111/j.1365-2109.1997.tb01316.x.
Dowd, P.F. 2000. Indirect reduction of ear molds and associated
mycotoxins in Bacillus thuringiensis corn under controlled and
open field conditions: utility and limitations. J. Econ. Entomol.
93(6): 1669–1679. doi:10.1603/0022-0493-93.6.1669.
European Food Safety Authority. 2006. Guidance document of the
Scientific Panel on Genetically Modified Organisms for the risk
assessment of genetically modified plants and derived food and
feed use. EFSA J. 374: 1–115. http://www.efsa.europa.eu/en/
scdocs/doc/374.pdf [accessed January 2009].
European Food Safety Authority. 2008. Safety and nutritional as-
sessment of GM plants and derived food and feed: the role of
animal feeding trials. Food Chem. Toxicol. 46(Suppl. 1): S1–
S70. doi:10.1016/j.fct.2008.02.013.
European Food Safety Authority. 2009. Statement of EFSA on the
consolidated presentation of opinions on the use of antibiotic re-
sistance genes as marker genes in genetically modified plants.
EFSA J. 1108: 1–8. http://www.efsa.europa.eu/en/scdocs/doc/
1108.pdf [accessed January 2010].
Einspanier, R., Klotz, A., Kraft, J., Aulrich, K., Poser, R.,
Schwa
¨gele, F., Jahreis, G., and Flachowsky, G. 2001. The fate
of forage plant DNA in farm animals: a collaborative case-study
investigating cattle and chicken fed recombinant plant material.
Eur. Food Res. Technol. 212(2): 129–134. doi:10.1007/
s002170000248.
Flachowsky, G., Chesson, A., and Aulrich, K. 2005. Animal nutri-
tion with feeds from genetically modified plants. Arch. Anim.
Nutr. 59(1): 1–40. doi:10.1080/17450390512331342368.
FAO. 2005. Review of the state of world marine fishery resources.
FAO Fish. Tech. Pap. 457. Food and Agriculture Organization
of the United Nations, Rome, Italy.
Francis, G., Makkar, H.P.S., and Becker, K. 2001. Antinutritional
factors present in plant-derived alternate fish feed ingredients
and their effects in fish. Aquaculture, 199(3–4): 197–227.
doi:10.1016/S0044-8486(01)00526-9.
Frøystad, M.K., Lilleeng, E., Bakke-Mckellep, A.M., Vekterud, K.,
Hemre, G.-I., and Krogdahl, A
˚. 2008. Gene expression in distal
intestine of Atlantic salmon (Salmo salar L.) fed genetically
modified soybean meal. Aquacult. Nutr. 14(3): 204–214. doi:10.
1111/j.1365-2095.2007.00519.x.
Frøystad-Saugen, M.K., Lilleeng, E., Bakke-McKellep, A.M.,
Vekterud, K., Valen, E.C., Hemre, G.I., and Krogdahl, A
˚. 2009.
Distal intestinal gene expression in Atlantic salmon (Salmo salar
L.) fed genetically modified maize. Aquacult. Nutr. 15(1): 104–
115. doi:10.1111/j.1365-2095.2008.00572.x.
Gatlin, D.M., Barrows, F.T., Brown, P., Dabrowski, K.,
Gaylord, T.G., Hardy, R.W., Herman, E., Hu, G., Krogdahl, A
˚.,
Nelson, R., Overturf, K., Rust, M., Sealey, W., Skonberg, D.,
Souza, E.J., Stone, D., Wilson, R., and Wurtele, E. 2007. Ex-
panding the utilization of sustainable plant products in aqua-
feeds: a review. Aquacult. Res. 38(6): 551–579. doi:10.1111/j.
1365-2109.2007.01704.x.
Gay, P.B., and Gillespie, S.H. 2005. Antibiotic resistance markers
in genetically modified plants: a risk to human health? Lancet
Infect. Dis. 5(10): 637–646. doi:10.1016/S1473-3099(05)70241-
3.
Gelvin, S.B. 2003. Agrobacterium-mediated plant transformation:
the biology behind the ‘‘gene-jockeying’’ tool. Microbiol. Mol.
Biol. Rev. 67(1): 16–37. doi:10.1128/MMBR.67.1.16-37.2003.
Gene Technology Act. 1993. [online.] Available from http://www.
lovdata.no/for/sf/dl/xd-19930820-0811.html [accessed 23 August
2010]. [In Norwegian.]
Glencross, B., Curnow, J., Hawkins, W., Kissil, G.W.M., and
Peterson, D. 2003. Evaluation of the feed value of a transgenic
strain of the narrow-leaf lupin (Lupinus angustifolius) in the diet
of the marine fish, Pagrus auratus. Aquacult. Nutr. 9(3): 197–
206. doi:10.1046/j.1365-2095.2003.00247.x.
Hammond, B.G., Vicini, J.L., Hartnell, G.F., Naylor, M.W.,
Knight, C.D., Robinson, E.H., Fuchs, R.L., and Padgette, S.R.
1996. The feeding value of soybeans fed to rats, chickens, cat-
fish and dairy cattle is not altered by genetic incorporation of
glysophate tolerance. J. Nutr. 126: 717–727.
Hansen, G., and Wright, M.S. 1999. Recent advances in the trans-
formation of plants. Trends Plant Sci. 4(6): 226–231. doi:10.
1016/S1360-1385(99)01412-0.
Harrison, L.A., Bailey, M.R., Naylor, M.W., Ream, J.E.,
Hammond, B.G., Nida, D.L., Burnette, B.L., Nickson, T.E.,
Mitsky, T.A., Taylor, M.L., Fuchs, R.L., and Padgette, S.R.
1996. The expressed protein in glyphosate-tolerant soybean, 5-
enolypyruvylshikimate-3-phosphate synthase from
Agrobacterium sp. strain CP4, is rapidly digested in vitro and is
not toxic to acutely gavaged mice. J. Nutr. 126: 728–740.
Hemre, G.I., Sanden, M., Bakke-Mckellep, A.M., Sagstad, A., and
Krogdahl, A. 2005. Growth, feed utilization and health of Atlan-
tic salmon Salmo salar L. fed genetically modified compared to
non-modified commercial hybrid soybeans. Aquacult. Nutr.
11(3): 157–167. doi:10.1111/j.1365-2095.2005.00328.x.
Hemre, G.I., Sagstad, A., Bakke-Mckellep, A.M., Danieli, A.,
Acierno, R., Maffia, M., Frøystad, M., Krogdahl, A
˚., and
Sanden, M. 2007. Nutritional, physiological, and histological re-
sponses in Atlantic salmon, Salmo salar L. fed diets with geneti-
cally modified maize. Aquacult. Nutr. 13(3): 186–199. doi:10.
1111/j.1365-2095.2007.00465.x.
Hohlweg, U., and Doerfler, W. 2001. On the fate of plant or other
foreign genes upon the uptake in food or after intramuscular in-
Sissener et al. 571
Published by NRC Research Press
Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by Institute of Marine Research (Norway) on 09/03/12
For personal use only.
jection in mice. Mol. Genet. Genomics, 265(2): 225–233.
doi:10.1007/s004380100450.
Hohn, B., Levy, A.A., and Puchta, H. 2001. Elimination of selec-
tion markers from transgenic plants. Curr. Opin. Biotechnol.
12(2): 139–143. doi:10.1016/S0958-1669(00)00188-9.
James, C. 2009. Global status of commercialized biotech/GM
crops: 2009. ISAAA, Ithaca, New York.
Jonas, D.A., Elmadfa, I., Engel, K.H., Heller, K.J.,
Kozianowski, G., Konig, A., Muller, D., Narbonne, J.F.,
Wackernagel, W., and Kleiner, J. 2001. Safety considerations of
DNA in food. Ann. Nutr. Metab. 45(6): 235–254. doi:10.1159/
000046734.
Kaushik, S.J., and Hemre, G.-I. 2008. Plant proteins as alternative
sources for fish feed and farmed fish quality. Chapter 12. In Im-
proving farmed fish quality and safety. Edited by Ø. Lie. Wood-
head Publishing Limited, CRC Press, Cambridge, UK. pp. 300–
319.
Kaushik, S.J., Cravedi, J.P., Lalles, J.P., Sumpter, J.,
Fauconneau, B., and Laroche, M. 1995. Partial or total replace-
ment of fish meal by soybean protein on growth, protein utiliza-
tion, potential estrogenic or antigenic effects, cholesterolemia
and flesh quality in rainbow trout, Oncorhynchus mykiss. Aqua-
culture, 133(3-4): 257–274. doi:10.1016/0044-8486(94)00403-B.
Kelly, A.M., and Green, C.C. 2006. Binding affinities of hepatic
nuclear estrogen receptors for genistein in Channel Catfish.
N. Am. J. Aquaculture, 68(2): 160–167. doi:10.1577/A05-081.1.
Knowles, B.H. 1994. Mechanism of action of Bacillus thuringiensis
insecticidal delta-endotoxins. Adv. Insect Physiol. 24: 275–308.
doi:10.1016/S0065-2806(08)60085-5.
Knudsen, I., and Poulsen, M. 2007. Comparative safety testing of
genetically modified foods in a 90-day rat feeding study design
allowing the distinction between primary and secondary effects
of the new genetic event. Regul. Toxicol. Pharmacol. 49(1):
53–62. doi:10.1016/j.yrtph.2007.07.003.
Kuiper, H.A., and Kleter, G.A. 2003. The scientific basis for risk
assessment and regulation of genetically modified foods. Trends
Food Sci. Technol. 14(5-8): 277–293. doi:10.1016/S0924-
2244(03)00067-0.
Kuiper, H.A., Kleter, G.A., Noteborn, H.P.J.M., and Kok, E.J.
2001. Assessment of the food safety issues related to genetically
modified foods. Plant J. 27(6): 503–528. doi:10.1046/j.1365-
313X.2001.01119.x.
Larkin, P., and Harrigan, G. 2007. Opportunities and surprises in
crops modified by transgenic technology: metabolic engineering
of benzylisoquinoline alkaloid, gossypol and lysine biosynthetic
pathways. Metabolomics, 3(3): 371–382. doi:10.1007/s11306-
007-0075-1.
Li, M.H., Hartnell, G.F., Robinson, E.H., Kronenberg, J.M.,
Healy, C.E., Oberle, D.F., and Hoberg, J.R. 2008. Evaluation of
cottonseed meal derived from genetically modified cotton as
feed ingredients for channel catfish, Ictalurus punctatus. Aqua-
cult. Nutr. 14(6): 490–498. doi:10.1111/j.1365-2095.2007.
00554.x.
Lucas, D.M., Taylor, M.L., Hartnell, G.F., Nemeth, M.A.,
Glenn, K.C., and Davis, S.W. 2007. Broiler performance and
carcass characteristics when fed diets containing lysine maize
(LY038 or LY038 MON 810), control, or conventional refer-
ence maize. Poult. Sci. 86: 2152–2161.
Mazza, R., Soave, M., Morlacchini, M., Piva, G., and Marocco, A.
2005. Assessing the transfer of genetically modified DNA from
feed to animal tissues. Transgenic Res. 14(5): 775–784. doi:10.
1007/s11248-005-0009-5.
McCann, M.C., Liu, K., Trujillo, W.A., and Dobert, R.C. 2005.
Glyphosate-tolerant soybeans remain compositionally equivalent
to conventional soybeans (Glycine max L.) during three years of
field testing. J. Agric. Food Chem. 53(13): 5331–5335. doi:10.
1021/jf0504317.
McLean, E., and Ash, R. 1987. The time-course of appearance and
net accumulation of horseradish-peroxidase (Hrp) presented or-
ally to rainbow trout Salmo Gairdneri (Richardson). Comp. Bio-
chem. Physiol. A, 88(3): 507–510. doi:10.1016/0300-9629(87)
90072-7.
McLean, E., Vondermeden, A.C., and Donaldson, E.M. 1990. Di-
rect and indirect evidence for polypeptide absorption by the tel-
eost gasterointestinal tract. J. Fish Biol. 36(4): 489–498. doi:10.
1111/j.1095-8649.1990.tb03551.x.
Molvig, L., Tabe, L.M., Eggum, B.O., Moore, A.E., Craig, S.,
Spencer, D., and Higgins, T.J. 1997. Enhanced methionine le-
vels and increased nutritive value of seeds of the transgenic lu-
pins (Lupinus angustifolius L.) expressing a sunflower seed
albumin gene. Proc. Natl. Acad. Sci. U.S.A. 94(16): 8393–8398.
doi:10.1073/pnas.94.16.8393.
Moreno-Fierros, L., Garcı
´a, N., Gutie
´rrez, R., Lo
´pez-Revilla, R.,
and Va
´zquez-Padro
´n, R.I. 2000. Intranasal, rectal and intraperi-
toneal immunization with protoxin Cry1Ac from Bacillus thurin-
giensis induces compartmentalized serum, intestinal, vaginal and
pulmonary immune responses in Balb/c mice. Microbes Infect.
2(8): 885–890. doi:10.1016/S1286-4579(00)00398-1.
Munkvold, G.P., Hellmich, R.L., and Showers, W.B. 1997. Re-
duced fusarium ear rot and symptomless infection in kernels of
maize genetically engineered for european corn borer resistance.
Phytopathology, 87(10): 1071–1077. doi:10.1094/PHYTO.1997.
87.10.1071.
Mu
¨ntz, K., Christov, V., Saalbach, G., Saalbach, I., Waddell, D.,
Pickardt, T., Schieder, O., and Wu
¨stenhagen, T. 1998. Genetic
engineering for high methionine grain legumes. Nahrung, 42:
125–127.
Nielsen, C. 2006. Tracing and characterisation of dietary DNA —
applications in Atlantic salmon. Ph.D. thesis, Deparment of
Biology, Faculty of Mathematics and Natural Sciences, Univer-
sity of Oslo, Norway.
Nielsen, C.R., Berdal, K.G., Bakke-McKellep, A.M., and Holst-
Jensen, A. 2005. Dietary DNA in blood and organs of Atlantic
salmon (Salmo salar L.). Eur. Food Res. Technol. 221(1–2): 1–
8. doi:10.1007/s00217-005-1160-1.
Padgette, S.R., Taylor, N.B., Nida, D.L., Bailey, M.R.,
MacDonald, J., Holden, L.R., and Fuchs, R.L. 1996. The com-
position of glyphosate-tolerant soybean seeds is equivalent to
that of conventional soybeans. J. Nutr. 126: 702–716.
Palka-Santini, M., Schwarz-Herzke, B., Ho
¨sel, M., Renz, D.,
Auerochs, S., Brondke, H., and Doerfler, W. 2003. The gastroin-
testinal tract as the portal of entry for foreign macromolecules:
fate of DNA and proteins. Mol. Genet. Genomics, 270: 201–
215. doi:10.1007/s00438-003-0907-2.
Papst, C., Utz, H.F., Melchinger, A.E., Eder, J., Magg, T.,
Klein, D., and Bohn, M. 2005. Mycotoxins produced by
Fusarium spp. in isogenic Bt vs. non-Bt maize hybrids under
European corn borer pressure. Agron. J. 97: 219–224.
Pelissero, C., Bennetau, B., Babin, P., Le Menn, F., and
Dunogues, J. 1991. The estrogenic activity of certain phytoestro-
gens in the siberian sturgeon Acipenser baeri. J. Steroid Bio-
chem. Mol. Biol. 38(3): 293–299. doi:10.1016/0960-0760(91)
90100-J.
Phipps, R.H., Deaville, E.R., and Maddison, B.C. 2003. Detection
of transgenic and endogenous plant DNA in rumen fluid, duode-
nal digesta, milk, blood, and faeces of lactating dairy cows.
J. Dairy Sci. 86(12): 4070–4078. doi:10.3168/jds.S0022-
0302(03)74019-3.
572 Can. J. Fish. Aquat. Sci. Vol. 68, 2011
Published by NRC Research Press
Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by Institute of Marine Research (Norway) on 09/03/12
For personal use only.
Prescott, V.E., Campbell, P.M., Moore, A., Mattes, J.,
Rothenberg, M.E., Foster, P.S., Higgins, T.J.V., and
Hogan, S.P. 2005. Transgenic expression of bean-amylase inhi-
bitor in peas results in altered structure and immunogenicity.
J. Agric. Food Chem. 53(23): 9023–9030. doi:10.1021/
jf050594v.
Ran, T., Mei, L., Lei, W., Aihua, L., Ru, H., and Jie, S. 2009. De-
tection of transgenic DNA in tilapias (Oreochromis niloticus,
GIFT strain) fed genetically modified soybeans (Roundup
Ready). Aquacult. Res. 40(12): 1350–1357. doi:10.1111/j.1365-
2109.2009.02187.x.
Reuter, T., and Aulrich, K. 2003. Investigations on genetically
modified maize (Bt-maize) in pig nutrition: fate of feed-ingested
foreign DNA in pig bodies. Eur. Food Res. Technol. 216: 185–
192.
Rombout, J., Lamers, C.H.J., Helfrich, M.H., Dekker, A., and
Tavernethiele, J.J. 1985. Uptake and transport of intact macro-
molecules in the intestinal epithelium of carp (Cyprinus carpio
L.) and the possible immunological implications. Cell Tissue
Res. 239(3): 519–530. doi:10.1007/BF00219230.
Sagstad, A., Sanden, M., Haugland, Ø., Hansen, A.-C.,
Olsvik, P.A., and Hemre, G.-I. 2007. Evaluation of stress- and
immune-response biomarkers in Atlantic salmon, Salmo
salar L., fed different levels of genetically modified maize (Bt
maize), compared with its near-isogenic parental line and a com-
mercial suprex maize. J. Fish Dis. 30(4): 201–212. doi:10.1111/
j.1365-2761.2007.00808.x.
Sagstad, A., Sanden, M., Krogdahl, A
˚., Bakke-McKellep, A.M.,
Frøystad, M., and Hemre, G.-I. 2008. Organs development,
gene expression and health of Atlantic salmon (Salmo salar L.)
fed genetically modified soybeans compared to the near-isogenic
non-modified parental line. Aquacult. Nutr. 14(6): 556–572.
doi:10.1111/j.1365-2095.2008.00630.x.
Sanden, M., Bruce, I.J., Rahman, M.A., and Hemre, G.I. 2004. The
fate of transgenic sequences present in genetically modified
plant products in fish feed, investigating the survival of GM
soybean DNA fragments during feeding trials in Atlantic sal-
mon, Salmo salar L. Aquaculture, 237(1–4): 391–405. doi:10.
1016/j.aquaculture.2004.04.004.
Sanden, M., Berntssen, M.H.G., Krogdahl, A
˚., Hemre, G.I., and
Bakke-McKellep, A.M. 2005. An examination of the intestinal
tract of Atlantic salmon, Salmo salar L., parr fed different vari-
eties of soy and maize. J. Fish Dis. 28(6): 317–330. doi:10.1111/
j.1365-2761.2005.00618.x.
Sanden, M., Krogdahl, A., Bakke-Mckellep, A.M.,
Buddington, R.K., and Hemre, G.I. 2006. Growth performance
and organ development in Atlantic salmon, Salmo salar L. parr
fed genetically modified (GM) soybean and maize. Aquacult.
Nutr. 12(1): 1–14. doi:10.1111/j.1365-2095.2006.00367.x.
Sanders, P.R., Lee, T.C., Groth, M.E., Astwood, J.D., and
Fuchs, R.L. 1998. Safety assessment of insect-protected corn. In
Biotechnology and safety assessment. 2nd ed. Edited by
J.A. Thomas. Taylor Francis, New York.
Saxena, D., and Stotzky, G. 2001. Bt corn has a higher lignin con-
tent than non-Bt corn. Am. J. Bot. 88(9): 1704–1706. doi:10.
2307/3558416.
Schubbert, R., Hohlweg, U., Renz, D., and Doerfler, W. 1998. On
the fate of orally ingested foreign DNA in mice: chromosomal
association and placental transmission to the fetus. Mol. Gen.
Genet. 259(6): 569–576. doi:10.1007/s004380050850.
Sire, M.F., and Vernier, J.M. 1992. Intestinal absorption of protein
in teleost fish. Comp. Biochem. Physiol. Physiol. 103(4): 771–
781. doi:10.1016/0300-9629(92)90180-X.
Sissener, N.H., Sanden, M., Bakke, A.M., Kroghdahl, A
˚., and
Hemre, G.I. 2009a. A long term trial with Atlantic salmon
(Salmo salar L.) fed genetically modified soy; focusing general
health and performance before, during and after the parr–smolt
transformation. Aquaculture, 294(1–2): 108–117. doi:10.1016/j.
aquaculture.2009.05.002.
Sissener, N.H., Bakke, A.M., Gu, J., Penn, M.H., Eie, E.,
Krogdahl, A
˚., Sanden, M., and Hemre, G.-I. 2009b. An assess-
ment of organ and intestinal histomorphology and cellular stress
response in Atlantic salmon (Salmo salar L.) fed genetically
modified Roundup Ready1soy. Aquaculture, 298: 101–110.
doi:10.1016/j.aquaculture.2009.10.011.
Sissener, N.H., Martin, S.A.M., Cash, P., Hevrøy, E.M.,
Sanden, M., and Hemre, G.I. 2010a. Proteomic profiling of liver
from Atlantic salmon (Salmo salar) fed genetically modified soy
compared to the near-isogenic non-GM line. Mar. Biotechnol.
12(3): 273–281. doi:10.1007/s10126-009-9214-1.
Sissener, N.H., Johannessen, L.E., Hevrøy, E.M., Wiik-
Nielsen, C.R., Berdal, K.G., Nordgreen, A., and Hemre, G.-I.
2010b. Zebrafish (Danio rerio) as a model for investigating the
safety of GM feed ingredients (soya and maize); performance,
stress response and uptake of dietary DNA sequences. Br. J.
Nutr. 103: 3–15. doi:10.1017/S0007114509991401.
Somers, D.A., and Makarevitch, I. 2004. Transgene integration in
plants: poking or patching holes in promiscuous genomes? Curr.
Opin. Biotechnol. 15(2): 126–131. doi:10.1016/j.copbio.2004.02.
007.
Suharman, I., Satoh, S., Haga, Y., Takeuchi, T., Endo, M.,
Hirono, I., and Aoki, T. 2009. Utilization of genetically modi-
fied soybean meal in Nile tilapia Oreochromis niloticus diets.
Fish. Sci. 75(4): 967–973. doi:10.1007/s12562-009-0106-0.
Sundell, K., Jutfelt, F., Agustsson, T., Olsen, R.E., Sandblom, E.,
Hansen, T., and Bjornsson, B.T. 2003. Intestinal transport me-
chanisms and plasma cortisol levels during normal and out-of-
season parr–smolt transformation of Atlantic salmon, Salmo
salar. Aquaculture, 222(1-4): 265–285. doi:10.1016/S0044-
8486(03)00127-3.
Sunilkumar, G., Campbell, L.M., Puckhaber, L., Stipanovic, R.D.,
and Rathore, K.S. 2006. Engineering cottonseed for use in hu-
man nutrition by tissue-specific reduction of toxic gossypol.
Proc. Natl. Acad. Sci. U.S.A. 103(48): 18054–18059. doi:10.
1073/pnas.0605389103.
Sveier, H., Norda
˚s, Berge, and Lied. 2001. Dietary inclusion of
crystalline D- and L-methionine: effects on growth, feed and pro-
tein utilization, and digestibility in small and large Atlantic sal-
mon (Salmo salar L.). Aquacult. Nutr. 7(3): 169–181. doi:10.
1046/j.1365-2095.2001.00169.x.
Taylor, N.B., Fuchs, R.L., MacDonald, J., Shariff, A.R., and
Padgette, S.R. 1999. Compositional analysis of glyphosate-
tolerant soybeans treated with glyphosate. J. Agric. Food Chem.
47(10): 4469–4473. doi:10.1021/jf990056g.
Ura
´n, P.A., Schrama, J.W., Jaafari, S., Baardsen, G.,
Rombout, J.H.W.M., Koppe, W., and Verreth, J.A.J. 2009. Var-
iation in commercial sources of soybean meal influences the se-
verity of enteritis in Atlantic salmon (Salmo salar L.). Aquacult.
Nutr. 15(5): 492–499. doi:10.1111/j.1365-2095.2008.00615.x.
Ursin, V.M. 2003. Modification of plant lipids for human health:
development of functional land-based omega-3 fatty acids.
J. Nutr. 133: 4271–4274.
van den Eede, G., Aarts, H.J., Buhk, H.J., Corthier, G., Flint, H.J.,
Hammes, W., Jacobsen, B., Midtvedt, T., van der Vossen, J.,
von Wright, A., Wackernagel, W., and Wilcks, A. 2004. The re-
levance of gene transfer to the safety of food and feed derived
from genetically modified (GM) plants. Food Chem. Toxicol.
42(7): 1127–1156. doi:10.1016/j.fct.2004.02.001.
Sissener et al. 573
Published by NRC Research Press
Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by Institute of Marine Research (Norway) on 09/03/12
For personal use only.
Van den Ingh, T.S.G.A.M., Krogdahl, A
˚., Olli, J.J.,
Hendriks, H.G.C.J.M., and Koninkx, J.G.J.F. 1991. Effects of
soybean-containing diets on the proximal and distal intestine in
Atlantic salmon (Salmo salar): a morphological study. Aquacul-
ture, 94(4): 297–305. doi:10.1016/0044-8486(91)90174-6.
Va
´zquez-Padro
´n, R.I., Moreno-Fierros, L., Neri-Baza
´n, L.,
de la Riva, G.A., and Lo
´pez-Revilla, R. 1999. Intragastric and
intraperitoneal administration of Cry1Ac protoxin from Bacillus
thuringiensis induces systemic and mucosal antibody responses
in mice. Life Sci. 64(21): 1897–1912. doi:10.1016/S0024-
3205(99)00136-8.
Va
´zquez-Padro
´n, R.I., Gonza
´les-Cabrera, J., Garcı
´a-Tovar, C.,
Neri-Bazan, L., Lope
´z-Revilla, R., Herna
´ndez, M., Moreno-
Fierro, L., and de la Riva, G.A. 2000. Cry1Ac protoxin from
Bacillus thuringiensis sp. kurstaki HD73 binds to surface pro-
teins in the mouse small intestine. Biochem. Biophys. Res.
Commun. 271(1): 54–58. doi:10.1006/bbrc.2000.2584.
Vlassov, V.V., Balakireva, L.A., and Yakubov, L.A. 1994. Trans-
port of oligonucleotides across natural and model membranes.
Biochem. Biophys. Acta Reviews on Biomembranes, 1197(2):
95–108. doi:10.1016/0304-4157(94)90001-9.
Watkins, K. 2009. Oilseeds of the future. Part 2. Inform, 20: 342–344.
Wilson, N.H., Hardisty, J.F., and Hayes, J.R. 2001. Short-term,
sub-chronic and chronic studies. In Principles and methods of
toxicology. 4th ed. Edited by A.W. Hayes. Taylor and Francis,
Philadelphia, Penn. pp. 917–957.
574 Can. J. Fish. Aquat. Sci. Vol. 68, 2011
Published by NRC Research Press
Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by Institute of Marine Research (Norway) on 09/03/12
For personal use only.
