Progress 09/01/05 to 08/31/09
Outputs
OUTPUTS: We developed datasets quantifying ecological and genetic risks posed by growth hormone (GH) transgenic Atlantic salmon. Objective 1 compared fitness of transgenic versus wild-type salmon. GH transgene expression showed no effects upon respiration of eggs, alevins and first-feeding fry, time to hatch, and proportion of yolk remaining near emergence. Cohabiting transgenic and control first-feeding fry did not differ in ability to compete for food and space. Growth rates of competing juveniles in hatchery or artificial stream environments showed significant effects of transgene, rearing density, feed ration, and environment. Transgenics did not experience reduced survival under low feed conditions. Transgenic juveniles matured as parr less frequently than non-transgenics, suggesting that transgene expression diverts energy toward growth and smoltification. Transgenic individuals had poor breeding performance in competitive breeding experiments among transgenic and wild adults. Competitive breeding experiments among transgenic and non-transgenic precocious parr indicated a substantially larger proportion of offspring were fathered by non-transgenic parr. The effects of salmonid community assemblages on relative growth and survival of transgenic and non-transgenic salmon suggested some capacity to influence relative expression fitness-related phenotypic traits. Transgenic hybrids between transgenic and non-transgenic Atlantic salmon and brown trout survived and showed accelerated growth rates, demonstrating a means for the transgene to spread to wild, heterospecific populations. Objective 2 quantified GH and IGF-I expression in transgenic and control salmon to determine mechanisms underlying rapid growth. Transgene expression began as early as eyed egg stage, months before enhancement of growth rate. GH receptors, myostatin, myostatin receptor, and growth factors MyoDI and MyoDII are all expressed in transgenic and non-transgenic salmon from pre-hatch to 300g juveniles. Quantitative RT-PCR indicated that expression of GH1 and GH2 receptor genes did not differ between transgenic and control salmon at early life stages. Transgene expression was examined at all stages in the life history of the salmon from fry to 4-7 kg adults. RT-PCR and northern blot analyses revealed transgene expression in all major body tissues. In all cases, expression was highest at the earliest life stages and declined as the fish aged/grew in size. An examination of the plasma GH and IGF levels in salmon from approximately 50g to 6 kg did not reveal significant differences between transgenics and controls. Objective 3 examined stress response of young GH transgenic, triploid, and control salmon to stresses of starvation, low dissolved oxygen (1.5-2.0 ppm), or no stress. Results of starvation and hypoxia experiments demonstrated that wild-type fish maintain homeostasis more effectively than transgenic fish, showing lower fluctuations in all measured hematological parameters. Work to be undertaken under objective 4 will utilize empirical data to predict the net fitness of GH-transgenic Atlantic salmon. PARTICIPANTS: Investigator Joe Brown died just before project activities commenced. Other project personnel made noteworthy contributions to execution of this project. Ph.D. candidate Darek Moreau executed much of the work pertaining to the ecological risk assessment. Other individuals contributing to that plan of work included K. Oake, A.H. Westley, D. Ings, A.K. Gamperl, J.A. Brown, and C. Conway. Participating in work pertaining to molecular characterization of transgene expression were R. Hobbs and M.J .King. Post-doctoral research associate Avner Cnaani played a leading role quantifying the response of transgenic salmon to stress. TARGET AUDIENCES: Key target audiences for this work include regulatory agencies (U.S. Food and Drug Administration, National Marine Fisheries Service, U.S. Fish and Wildlife Service, U.S. Department of Agriculture, state departments of fisheries and marine resources, Fisheries and Oceans Canada, other national and international agencies), scientific investigators, the non-governmental organization sector, and the general public. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The investigators have interacted with AquaBounty, producer of the transgenic Atlantic salmon. Coming to appreciate the potential hazards posed by transgenic Atlantic salmon in natural ecosystems, the management team at AquaBounty has committed to produce the fish only under conditions of strict confinement. This commitment may have bearing on the U.S. Food and Drug Administration's (FDA) deliberation on whether and under what conditions to approve commercial production of this genetically modified product. The investigators have interacted with FDA officials about transgenic salmon and transgenic animals more generally, for example in the lead up to FDA's recent publication of its policy for oversight of genetically engineered animals for food. Publication of findings from this project will provide critical empirical science to inform policy and decision making and influence public perception regarding genetically engineered aquatic animals. This research has facilitated training of one postdoctoral fellow, one Ph.D. candidate, one M.S. candidate, one undergraduate, and two high school students. The junior-level students have gained valuable research experience to help decide a career path. The graduate-level students have received training uniquely positioning them to contribute to the field of biotechnology and related sciences, whether in research, teaching, policy or regulatory capacities.
Publications
- Oake, K., P. A. H. Westley , D. T. R. Moreau and I. A. Fleming. 2009. Competition, growth and survival of growth hormone transgenic and non-transgenic Atlantic salmon (Salmo salar) and Atlantic salmon-brown trout hybrids (S. salar trutta) in naturalized stream environments. In preparation.
- Moreau, D. T. R., P. A. H. Westley and I. A. Fleming. 2009. Interspecific hybridisation as a potential route for the introgression of transgenes into wild populations: the case of Atlantic salmon (Salmo salar) and brown trout (Salmo trutta). In preparation.
- Fleming, I. A., D.T.R Moreau, and D. Ings. 2009. The effect of stream salmonid community assemblages on the growth, survival and energy use of transgenic and non-transgenic Atlantic salmon (Salmo salar). In preparation.
- Moreau, D. T. R. and I. A. Fleming. 2009. The interaction of transgenic and non-transgenic Atlantic salmon (Salmo salar) siblings across multiple environments. In preparation.
- Moreau, D. T. R. and I. A. Fleming. 2009. The reproductive performance of growth hormone transgenic Atlantic salmon (Salmo salar) males. In preparation. Moreau, D. T. R. and I. A. Fleming. 2009. Reproductive life history characteristics of growth hormone transgenic Atlantic salmon (Salmo salar) precocious male parr. In preparation.
- Moreau, D. T. R., A. K. Gamperl, G. L. Fletcher, and I. A. Fleming. 2009. Rates of development and metabolism of growth hormone transgenic Atlantic salmon (Salmo salar) during early ontogeny. In preparation.
- Moreau, D. T. R., J. A. Brown, G. L. Fletcher and I. A. Fleming. 2009. Competition between growth hormone transgenic and non-transgenic first-feeding Atlantic salmon fry and their relative performance in low feed stream microcosms. In review.
- Moreau, D. T. R. and I. A. Fleming. 2009. The potential ecological and genetic impacts of transgenic aquaculture biotechnologies: Eco-evolutionary considerations for managing the blue revolution. In: Aquaculture Biotechnology. Fletcher, G. L., Rise, M. L. editors. Blackwell Publishing Inc. In review.
- Moreau, D.T.R., R. Hobbs, A. Cnaani, I. Fleming, G. Fletcher, E. Mclean, and E. Hallerman. 2009. Environmental risk assessment parameters for growth hormone transgenic Atlantic salmon, Salmo salar. Transgenic Research 18: in press.
- Moreau, D.T.R., G.L. Fletcher and I.A. Fleming. 2009. Rates of maturation and reproductive performance in growth hormone transgenic Atlantic salmon precocious male parr. Canadian Society of Ecology and Evolution Conference 2009, Halifax, Nova Scotia, Canada. May 14-17, 2009.
|
Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: Our goal is to develop datasets quantifying ecological and genetic risks posed by growth hormone (GH) transgenic Atlantic salmon. Objective 1 quantifies fitness. During 2007, transgenic and non-transgenic 0+ parr were used in competitive breeding experiments. We collected behavioral data and are conducting microsatellite analyses to determine paternity. High rates of precocious maturation were found among both transgenic and non-transgenic age 1+ parr. We are comparing the development, growth and survival of transgenic and non-transgenic crosses with brown trout, a hybrid cross that occurs in nature, providing a means for transgene spread to wild populations. Stream ecosystems in which Atlantic salmon are native comprise a community of salmonid species including brook charr and brown trout. During 2008, we executed an experiment testing effects of salmonid community assemblages on relative growth and survival of transgenic and non-transgenic salmon; these data are being assessed. Objective 2 quantifies dynamics of circulating GH and IGF-I in transgenic and control salmon to explain mechanisms underlying rapid growth. We have completed study of EO-1α GH transgene expression through the life cycle. Transgene expression begins as early as eyed egg stage, several months before enhancement of growth rate. Therefore, delay in growth acceleration is not due to lack of GH transgene expression. We examined expression of GH receptors, myostatin genes, myostatin receptor, and growth factors MyoDI and MyoDII using RT-PCR. All these genes are expressed in transgenic and non-transgenic salmon from pre-hatch embryos to 300g juveniles. However, expression of GH receptor genes was greater in transgenics and increased during early development. Expression of myostatin genes in both transgenics and controls increased with size/age. A more detailed study of expression of myostatin, GH receptor and GH transgenes using quantitative RT-PCR will be completed by spring 2009. We have collected blood plasma samples from all size-classes of transgenic and non-transgenic salmon and shipped them to Dr. Thrandur Bjornsson (University of Gothenburg Sweden), who is collaborating on GH and IGF analysis. Objective 3 examines innate immune response of young GH transgenic salmon to stress. The American collaborators traveled to Canada to conduct challenge tests. Groups of GH-transgenic, triploid, and wild-type Atlantic salmon were subjected to one-week starvation, low dissolved oxygen (1.5-2.0 ppm), or no stress in triplicated tanks. Blood samples taken from anaesthetized fish 1, 3, 6, or 24 hours after stress were analyzed using a radiometer. In unstressed salmon, parameters did not differ among genotypes, except that blood pH was higher and potassium levels lower in wild-type than in triploid or transgenic salmon. Results of starvation and hypoxia experiments demonstrated that wild-type fish maintain homeostasis more effectively than transgenic fish, showing lower fluctuations in all measured hematological parameters. Results of the time-course study imply that transgenic fish are more stressed than wild-type fish, as reflected in higher and faster response. PARTICIPANTS: This research has facilitated training of one postdoctoral fellow, one Ph.D. candidate, one M.S. candidate, one undergraduate, and two high school students. The junior-level students have gained valuable research experience to help decide a career path. The graduate-level students have received training uniquely positioning them to contribute to the field of biotechnology and related sciences, whether in research, teaching, policy or regulatory capacities. TARGET AUDIENCES: Key target audiences include: - other scientists - we made a presentation at a recent biotechnology and fisheries science conferences, - aquaculturists - we made a presentation at a recent aquaculture conference, and - general public - the principal investigator has spoken with reporters from approximately six media outlets during the reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
As these data become available through publication, they will provide critical empirical science to inform policy and decision making and influence public perception regarding genetically engineered aquatic animals. For example, this research gave the P.I. visibility in the field, so that when the U.S. Food and Drug Administration recently published its draft policy for oversight of genetically engineered animals for food, he was interviewed by leading media outlets.
Publications
- Moreau, D. 2008. An ecological assessment of growth hormone transgenic Atlantic salmon. 18th Annual APICS Aquaculture Conference, St. Johns, Newfoundland, Canada, March 7-9, 2008.
- Moreau, D. T. R., C. Conway, D. W. Ings, G. L. Fletcher and I. A. Fleming. 2008. The interaction of transgenic and non-transgenic Atlantic salmon siblings across multiple environments. 61st Meeting of the Canadian Conference for Fisheries Research, Halifax, Nova Scotia, Canada, January 3-5, 2008.
- Moreau, D. T. R., R. Hobbs, A. Cnaani, I. Fleming, G. Fletcher, E. Mclean, and E. Hallerman. 2007. Environmental risk assessment parameters for growth hormone transgenic Atlantic salmon, Salmo salar. Transgenic Research 16: 851.
|
Progress 09/01/06 to 08/31/07
Outputs
OUTPUTS: Our goal is to develop empirical data quantifying ecological and genetic risks posed by growth hormone (GH) transgenic Atlantic salmon. Objective 1 regards survival and reproductive components of fitness. Analyses of embryological development and larval survival are completed. Experiments initiated in 2007 assess early viability and territoriality of GH transgenic salmon relative to wild individuals. Experiment 2c investigates the effects of genotype (+/- of transgene and family of origin) and environment (feed level and relatedness of conspecifics) on performance of parr. Experiment 2d examines the effect of kinship and density-induced competition on survival and growth in a semi-natural environment. Feed quantity, feeding method, family control and age of fish differ from last year, investigating effects of onset of phenotypic expression of the transgene on survival. These experiments and molecular analyses are accomplished. During fall 2006, breeding performance of
captive-reared transgenic and wild anadromous salmon was compared over nine trials. When directly competing for access to spawning females, wild males spawned 18 times, while only a single transgenic male spawned once. The few 0+ transgenic male parr that matured in fall 2006 did so late in the breeding season, and hence were not used for experimentation. We are preparing for the 2007 breeding season. Low maturation rates may be due to low feed ration during rearing. While transgenics were within the size range common for early-maturing parr, the greater metabolic demands associated with GH transgenesis may prevent allocation of energy to maturation. A study is ongoing to address this hypothesis and provide mature transgenic parr for breeding experiments. Individuals from 8 hemizygous-crossed families were distributed into tanks with high or low feeding. If this study produces mature 0+ transgenic male parr, their breeding performance will be compared to that of 1+ non-transgenic
parr. Objective 2 compares levels of circulating GH and IGF-I in transgenic and control salmon. Semi-quantitative RT-PCR analysis of transgene expression through the growth of transgenic salmon was expanded to include intestinal tissue. To determine plasma GH and IGF-I levels, we are collecting plasma from a range of salmon size-classes. The analyses will be carried out by collaborator Thrandur Bjornsson in 2008. We are examining molecular factors - growth hormone receptors, myostatin, myostatin receptors, MyoDI and MyoDII, myogenin - that may be responsible for the rapid growth phenotype. We focus on muscle tissue because it accounts for over 50% of body mass. RT-PCR showed that all these factors are expressed from prehatch embryos to post smolt-sized salmon. The intensity of PCR amplicons for some factors suggests how expression changes with age. Results will be examined more closely using quantitative RT-PCR. We expect to complete analyses early in 2008. Objective 3 examines innate
immune response of young GH transgenic salmon to stress and immunological challenge. The American collaborators are in Canada at this writing to conduct challenge tests and collect samples.
TARGET AUDIENCES: As noted above, P.I. Eric Hallerman made a progress report-type presentation at a relevant research conference and spoke informally with key stakeholders in the private (AquaBounty) and public (U.S. Food and Drug Administration, Fisheries and Oceans Canada) sectors.
Impacts
P.I. Eric Hallerman made a progress report-type presentation, "Environmental risk assessment parameters for growth hormone transgenic Atlantic salmon", at the Transgenic Animal Research Conference held at Lake Tahoe, CA on August 13-15, 2007. While there, he spoke informally with representatives of AquaBounty (the company producing the salmon), the U.S. food and Drug Administration, and Fisheries and Oceans Canada.
Publications
- Hallerman, E.M., E. McLean, and I.A. Fleming. 2007. Effects of growth hormone transgenes on the behavior and welfare of aquacultured fishes: a review identifying research needs. Applied Animal Behavior Science 104:265-294.
- Hobbs, R.S. and G.L. Fletcher. 2007. Tissue specific expression of antifreeze protein and growth hormone transgenes driven by the ocean pout (Macrozoarces americanus) antifreeze protein OP5a gene promoter in Atlantic salmon (Salmo salar). Transgenic Research DOI 10.1007/s11248-007-9128-5
|
Progress 09/01/05 to 09/01/06
Outputs
Empirical estimation of fitness parameters for growth hormone (GH) transgenic Atlantic salmon (Salmo salar) in near-wild systems is lacking, a knowledge gap that is particularly notable given that the line examined in this research is the subject of an application to FDA for approval for commercial production. The goal of our research is to develop empirical data useful for quantifying ecological and genetic risks posed by GH transgenic Atlantic salmon in the wild. Our first objective is to quantify key aspects of the survival and reproductive components of fitness of GH transgenic Atlantic salmon in near-natural systems. Oxygen consumption data did not show a difference in the routine metabolism of transgenic and non-transgenic eyed-eggs and alevins. Preliminary data on development also suggest no difference between transgenic and non-transgenic individuals. Our data indicate that the transgene does not influence survival up to emergence from the gravel, although
developmental data from replicate families are required for conclusive evidence. Experiments with newly-emerged, first-feeding fry did not indicate transgene-induced behavioral differences. Pair-wise dominance trials resulted in the classic intruder-resident relationship, uninfluenced by the transgenic genotype of individuals. Similarly, growth and survival in the artificial stream study did not differ between transgenic and non-transgenic individuals at the two density treatments, but density influenced growth rate such that fry lost less mass at low density. The loss of mass observed in this study was not expected, although it is entirely plausible under food-limited environmental conditions. We are beginning experiments to address the reproductive component of fitness. Our second objective is to compare levels of circulating GH and insukin-like growth factor I (IGF-1) in transgenic and control fish. Study of adult salmon that were hemizygous for the EO-1α transgene revealed,
with the use of RT-PCR, that the GH transgene was expressed in most body tissues (heart, intestine, spleen, liver, kidney, stomach, ovary, gill filaments, muscle, skin, brain), with the exception of blood cells. However, northern blot analysis indicated significant levels of expression were confined to the spleen and pituitary. Using RT-PCR procedures, we found that expression of the GH transgene in hemizygous transgenic salmon was initiated as early as the eyed egg stage of development and continued throughout the life of the salmon. Preliminary work on IGF-I expression showed that there do not appear to be differences in expression between transgenics and control salmon.
Impacts
This line of GH transgenic Atlantic salmon is the subject of a request before the U.S. Food and Drug Administration for approval for commercial production. However, risk assessment is constrained by lack of empirical data on fitness of transgenic Atlantic salmon in the wild. This proposed research will result in an empirical data base on fitness of GH transgenic Atlantic salmon, with key survival and reproductive fitness components estimated in a near-wild system, fitness traits including early viability, age at maturity, mating success, and male fertility. Our preliminary data reported here do not suggest dramatic impacts of transgene expression on early survival and competitive ability of Atlantic salmon. However, replication of these experiments and extension of observations over the life and reproductive activity of the fish will be needed before firm inferences can be reached on the ecological and genetic impacts of transgene expression. Our data ultimately will
be input into a predictive net fitness model to identify which factors may warrant further research. Subsequent experiments will quantify components of net fitness over the entire life cycle. Our findings will prove useful for supporting regulatory decision-making regarding whether and under what conditions this transgenic line might be approved for commercial use. More generally, our findings will advance knowledge supporting risk assessment for genetically modified aquatic organisms, and on how physiological processes affect behavior and resource competition.
Publications
- Hallerman, E.M., E. McLean, and I.A. Fleming. 2006. Effects of growth hormone transgenes on the behavior and welfare of aquacultured fishes: a review identifying research needs. Applied Animal Behaviour Science, in press.
|
Progress 10/01/04 to 09/30/05
Outputs
We are assessing ecological and genetic risks posed by Atlantic salmon expressing a growth hormone (GH) transgene in a stream mesocosm and in the laboratory. To assess the fitness of transgenic fish and their progeny in the wild, we will quantify development rate of non-transgenic and transgenic embryos from fertilization until feeding, metabolic rate of non-transgenic and transgenic embryos at eyed-egg stage, when oxygen demand peaks, and survival differences between non-transgenic and transgenic embryos through eyed-egg stage under different levels of dissolved oxygen. We will quantify survival following emergence, a stage determining recruitment to the population. We will assess the effect of GH transgenesis on early male maturation under three growth regimes determined by food and temperature. We will quantify the reproductive success of early-maturing males of transgenic and non-transgenic origin in competition for breeding females in a stream mesocosm. We will
investigate GH transgene and IGF-I expression from larvae to maturity to determine whether there is tissue specificity regarding transgene transcription and blood plasma GH and IGF-I levels. We will determine whether transgenic salmon are compromised immunologically following thermal shock or fasting by tracking plasma protein concentration, hematocrit, and acute phase reaction proteins (lysozyme, ceruloplasmin and haptoglobin). We will use our empirical data and an existing net fitness model to predict the net fitness of GH transgenic salmon and transgene fate in near-natural ecosystems. Because this study is only now underway, our progress encompasses detailed planning of matings, set-up of project infrastructure, and search for graduate research assistants.
Impacts
This line of GH transgenic Atlantic salmon is the subject of a request before the U.S. Food and Drug Administration for approval for commercial production. However, risk assessment is constrained by lack of empirical data on fitness of transgenic Atlantic salmon in the wild. This proposed research will result in an empirical data base on fitness of GH transgenic Atlantic salmon, with key survival and reproductive fitness components estimated in a near-wild system, fitness traits including early viability, age at maturity, mating success, and male fertility. Our data will be input into a predictive model, the net fitness model of Muir and Howard, identifying which factors may warrant further research. Subsequent experiments will quantify components of net fitness over the entire life cycle. Our findings will prove useful for supporting regulatory decision-making regarding whether and under what conditions this transgenic line might be approved for commercial sue. More
generally, our findings will advance knowledge supporting risk assessment for genetically modified aquatic organisms, and on how physiological processes affect behavior and resource competition.
Publications
- No publications reported this period
|
|