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Apply Modern Biotechnology…
• To better understand and manage natural populations:– Molecular genetic tools– Genomics
• To modify or manipulate organisms:– Repro-technologies– Cloning– Genetic engineering
• To determine effects of modified organisms on natural populations
Expect combinations of these
Understand and Manage Natural PopulationsMany advantages over older methodologies
• Molecular genetic tools– Conservation genetics – Forensics– Pathology– Monitor effects of introduced organisms
• Genomics– Understand gene function– Marker-assisted selection– Monitor for effects of pollutants, environmental change
Modify or Manipulate Organisms• Repro-technologies
– *Chromosome set (ploidy) manipulations– *Cryopreservation of gametes/embryos– Gynogenesis/Androgenesis– Nuclear transplantation, embryo transfer, etc.
• Cloning – propagate endangered species– Somatic cell – e.g., Guar– Primordial cells –e.g. r. trout in Masu salmon (Nature 8/5/04)
– Embryonic stem cell• Genetic Engineering – recombinant DNA
– *modify performance traits – microbes, plants, animals– control invasive species– Vaccines
* Already some large-scale uses
Oilseed rape not shown. Data from James 2001 (Int’l Serv. for the Acquisition of Agri-biotech Appl. (ISAAA) and USDA NASS April 2004.
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50
60
70
80
90
1996 1997 1998 1999 2000 2001 2002 2003
Perc
ent A
dopt
ion
Soybeans
Cotton
Corn
Bt Corn
Crop GEOs In Use
Slide from K. Oberhauser
Examples of Plant GEOs: Field test to Release
Trait/ structural gene SpeciesDrought and salt tolerance Protein/enzyme genes from bacteria or other plants
turfgrasses: Bermudagrass, creeping bentgrass, Kentucky bluegrass, perennial ryegrasses
Herbicide tolerance, some with altered growth or disease resistance CBI or enzyme EPSPS
turfgrasses, poplar, cottonwood, Eucalyptus, sweetgum, wheat
Insect resistance specific Bt endotoxin (among many)
Loblolly pine, poplar, spruce, cranberry
Disease resistanceVirus coat protein, various antibacterial or antifungal genes
papaya*, plum ∆, apple, pear, grape
*Deregulated/Commercialized ∆Release permit CBI = Confidential Business Information
NRC 2004. Biological Confinement of Genetically Engineered Organisms. http://books.nap.edu
Plant GEOs: Research to Field Tests
Trait/ structural gene SpeciesDecreased lignin contentSpecific enzymes from bacteria or poplar; ligase antisense gene from poplar; or CBI
poplar #, pine #, turfgrass (Paspalum notatum) #
BioremediationMercuric ion reductase (from E. coli), cytochrome P450 (from human)
poplar#
Pharmaceuticals, biologics, industrial chemicalsswine viral vaccine, avidin, trypsin, high laurate many others in research
corn - swine vaccine #, avidin**, trypsin *, canola - laurate *numerous other crops
Rehabilitate endangered speciesFungal blight resistance genes from Asian chestnut
American chestnut
# Field test *Commercialized **Commercialized via field test notification
NRC 2004 Biological Confinement of Genetically Engineered Organisms. Snow et al. 2004 Ecol. Soc. Am. GEO Position Statement. Nature Biotechnology 2004 Editorial.
www.glofish.com
GloFish casts light on murkypolicing of transgenic animals
Nature 27 November 2003
The New York Times Nov 22, 2003Gene-Altering RevolutionNears the Pet Store:Glow-in-the-Dark Fish
First Transgenic Animal on U.S. Market
Marketed without regulatory environmental review. FDA is lead authority.
Could become first transgenic animal approved for large-scale farming and human food
The New York Times
Age = 7.5 months
Transgenic = 1.2 Kg., Unmodified = 200 g
Http://webhost.avin.net/afprotein/peidof.htm
Engineered with ocean pout antifreeze gene promoter + chinook salmon growth hormone gene
Novel proteins or novel gene regulation alter physiology. Alter ecological roles?
(Devlin et al. 1994)
Growth hormone expressed in cold waters & unlinked from seasonal temp. cue.
Smoltification precocious.
Age =14 monthsLargest transgenic = 41.8 cm
Age = 6 months; autotransgenic = 255.4 g (Nam et al. 2001)
Auto-transgenic mud loach: β-actin promoter linked to GH gene. Growth increase >30 fold.Gigantism.
Aquatic GEOs in the PipelineMarine Biotechnology Briefs http://www.fw.umn.edu/isees/MarineBrief
Enhanced growth & food conversion growth hormone genes
Atlantic salmon*, coho salmon, common carp#, mud loach, Nile tilapia#, rainbow trout*
Disease resistance mammalian interferon moth or porcine antibacterial cecropin B
grass carp, channel catfish, medaka
Cold water tolerance ocean pout antifreeze protein
goldfish
Carbohydrate digestibility mammalian enzymes for glucose metabolism
rainbow trout
Trait / structural gene Species
*applying for approval: U.S., Canada. # preparing to apply: Cuba, P.R. China
Aquatic GEOs in the PipelineMarine Biotechnology Briefs http://www.fw.umn.edu/isees/MarineBrief
Bioremediation chicken metallothionein (heavy metals binding)
unicellular algae (Chlamydomonas)
Thrive in absence of light human glucose transporters
microalgae – normally obligate photoautotroph
Secrete pharmaceutical human clotting factor VII
Nile tilapia
Retroviral vectors with marker genes step towards engineering production traits
live bearing fish (Poeciliopsis), crustacean (crayfish), mollusk (surfclam)
Trait / structural gene Species
• Various genetic engineering methods – 2 examples– Sex Ratio Distortion – daughterless carp technology– Engineered fitness disadvantage – site-specific selfish gene
• Feasibility study for FWS – genetic biocontrol of invasive fish in Gila River Basin, AZ, focus on green sunfish, red shiner, mosquito fish (Kapuscinski et al., ongoing)
Deleterious transgene spread to control invasive fish species
www.marine.csiro.au/LeafletsFolder/pdfsheets/Daughterless_carp_thirteenmay02.pdf
Mallee ResearchStation
4 nights’ catch, 1917Lascelles Victoria
Genetic Biocontrol House mouse plagues
Slide from Tony Peacock
Virally-vectored immunocontraception
Isolate ZP3 DNA
Insert DNA into mouse-specific virus
(recMCMV)
Infect mice with recMCMV
The mouse’s immune system blocks reproduction
The egg protein ZP3 is essential for reproduction
Slide from Tony Peacock
Risk Assessment and Management
• General agreement on case-by-case approach for GEOs
• Environmental biosafety science develops methodologies and generates empirical data needed for scientifically reliable risk assessment and management
• Strategies to cope with limits to prediction
Systematic Risk Assessment1. Identify hazard - what event posing harmful
consequences could occur? [knowledge is best here]
2. Estimate exposure - how likely is the hazard? [ability varies case-by-case; e.g. lack confirmed methodology for fish]
3. Predict harms & severity - what would be harms and how bad are they? [ability varies; need confirmed methodology]
4. Estimate risk –likelihood versus severity of harm [limits to quantification; depends on prior steps]
Kapuscinski 2002. Controversies in Designing Useful Ecological Assessments….National Research Council (NRC) 2004. Biological Confinement of Genetically Engineered Organism
Systematic Risk ManagementRisk reduction - what can be done to reduce
likelihood or mitigate consequences of harm? [Focus has been on confinement – see NRC 2004]
Post-release monitoring* - how effective are risk reduction actions? [Little attention so far]
Remedial action - What corrective action if monitoring findings are unacceptable? [Largely ignored so far]
*only way to learn and improve future decisions (Adaptive Management)
Kapuscinski 2002; NRC 2004
1. Identify potential hazards
• Gene flow to related taxa (interbreeding)• Invasion by alien species (is GEO more invasive than
unmodified?)• Interact with non-target organism• Evolution of resistance (pesticide-producing GEO)• Changes in viral disease (virus-resistant GEO)• Horizontal gene flow (1arily microorganisms)
Scientists’ Working Group on Biosafety 1998. www.edmonds-institute.org/manual.htmlPew Initiative on Food and Biotechnology 2003National Research Council 2004Ecological Society of America. 2004
2. Estimate exposure to hazard• Need a confirmed methodology involving
tractable and repeatable tests that can be conducted in confined settings
• Don’t have this yet but…
• Net fitness methodology is one promising candidate
(Net fitness methodology: Muir and Howard 2001, 2002)
wild-type medaka
transgenic medaka
Photos: Mike Morton
top view
Can we confirm the methodology? Ongoing test …
side views
Kapuscinski laboratory
0.0000
0.0250
0.0500
0.0750
0.1000
0.1250
400 (a)400 (b)
400 (c) 67 (a)
67 (b)67 (c)
treatment (sGH line number)
tran
sgen
e fr
eque
ncy
initial freq
final freq
Control x 3 replicates
1st GEO line x 3 replicates
2nd GEO line x 3 replicates
unmodified population (N = 353)
Released 20 pMTsGH-400 into unmodified (N=353)
Released 20 pMTsGH-67 into unmodified (N=353)
1st trial: transgene fate after 2 generations; population size equal across all treatments at end of trial.
(2nd trial currently underway.)
[Method predicts transgene spread] [Method predicts
transgene spread, then population decline]
3. Predict Harms and Severity
• Examples for fish and wildlife:1. Loss of unique genetic resources – e.g., center of
origin, extinction by hybridization2. Decline in abundance of species of special
concern - target of fishing/hunting, endangered, keystone in food web, culturally important, etc.
3. Decline in resilience of biological community—ability to recover from external disturbances
Challenges: cumulative, long-term, large-scaleScientists’ Working Group on Biosafety 1998. Pew Initiative on Food and Biotechnology 2003
Predicting harms: transgenic fish for aquaculture
(photo: Devlin et al. 1994)
• Guidance based on literature syntheses, but few GEO studies
• Lab study of one line of growth-hormone transgenic coho salmon (Devlin et al. 2004) – relevance to field conditions?
High food availability: transgenics did not competitively interfere with growth of nontransgenics.
Low food availability: populations with transgenics crashed and those without continued to increase in biomass.
UCS 2002. Pharm and Industrial Crops
For pharma/industrial crops:• Potential harm to wildlife feeding on plants / seeds• Potential harm to ecological resilience
– via exposure of pollinators, herbivores, soil inhabitants– via transgene spread to wild/weedy relatives– via bioaccumulation
Industry Projections:Market of $100s billions by 2010
Hazard Potential HarmDensity-dependent compensation for X years
Wipe out endangered fish before biocontrol effect prevails
Failure in intended trait change Increased number of fit non-natives increases disruption of native fish
Transgene side effect on trait that enhances predation or competition
Increases disruption of native fish before biocontrol effect prevails
Transgene spread to native range of species
Depress or extirpate native populations
Transgenic fish caught for eating Harm to human health
Horizontal gene transfer to non-target species (very hypothetical)
Depress populations of non-target species
Transgenic Fish for BiocontrolD
ecre
asin
g lik
elih
ood
(in g
ener
al)
Kapuscinski et al. In preparation
Photo: Nick Didlick
Holmlund & Hammer (1999)
•Individual species
•Equilibrium
•Linear – gradual change
•PREDICTABLE
•Socio-ecological system
•Multiple states•Non-linear – can flip to new state
•EXPECT SURPRISE
Prevailing Framework
Emerging Framework
Functional homogenization reduces resilience
Composition of, variation in, and spatial distribution of traits of the species in a community.
A-C: historical communities.a-c: homogenized communities.
External shock
Olden et al. (2004)
Biotechnology - Prevailing Approach
Small polygon – policy decisionGreen dotted arrow – ad-hoc learning
Expert PrioritySetting
BenefitsAssessment
Field Test ofDesired Traits
RiskAssessment
andManagement
Ad-HocDetection of
Problems
CorrectiveActions
FieldTesting
Decision
Commer-cializationDecision
PolicyEvaluation& Review
ResearchFundingDecision
TIMELINE FOR PREVAILING RESEARCH, DEVELOPMENT AND APPLICATION
Research &Development
Studies
RiskAssessment of
Proposed Field Test
Deliberation usually entails public review just before or after decision
Biotechnology - Pro-Active Approach
Small polygon – policy decision. Solid green arrow – adaptive learning. Italics – pro-active steps
ParticipatoryPriority Setting
&Safety Criteria
Setting
ProspectiveBenefits
Assessment
ProspectiveRisk
Assessment
Safety Design
BenefitsAssessment
Field Test ofDesired Trait
& Safety
SafetyManagement
ParticipatoryBenefits &
SafetyMonitoring
and Follow-up
Long-term,Large Scale
Studies
FieldTesting
Decision
Commer-cializationDecision
PolicyEvaluation& Review
ResearchFundingDecision
TIMELINE FOR PROACTIVE RESEARCH, DEVELOPMENT AND APPLICATION
Research &Development
Studies
SafetyTesting
RiskAssessment of
Proposed Field Test
Kapuscinski et al. 2003. Nature Biotechnology
Deliberation is linked to analysis from the outset
Prevailing approach – little steering to be safer from outset
Ris
k (li
kelih
ood
of h
arm
)High
Low
Severity of HarmLow High
NRC 2004. Miller et al. 2002
Ris
k (L
ikel
ihoo
d of
Har
m)
Frequent
Very closeto never
Severity of Harm
None As bad asit can get
Likelihood should decrease asseverity of harm increases.
‘Safety first’: safety criteria to impose upper limit to risk
© ISEES & S. Hann 2003
As bad asit can get
Verylarge
Sig-nificant
SmallNone
Severity of Harm(Includes Cumulative Effects)
Frequent
Common
Rare
Very closeto never
Very unlikely
Max
imum
Acc
epta
ble
Like
lihoo
d of
Har
m
Safeenough
Not safeenough
© ISEES & S. Hann 2003
Example of Deliberation Point: Effectof Benefits on Upper limit of
Acceptable Risk
As bad asit can get
Verylarge
Sig-nificant
SmallNone
Severity of Harm
Frequent
Common
Rare
Very closeto never
Very unlikely
Max
imum
Acc
epta
ble
Like
lihoo
d of
Har
m
IncreasingBenefits
DecreasingBenefits
© ISEES & S. Hann 2003
Pro-active Australian approach:Genetic biocontrol of invasive fish
• Will the genetic method work?– Under real conditions– Credible evidence before deployment
• What are the risks?– Environmental– Human health
• Answer via multi-prong program– Progress from simple to more complex
tests of efficacy and potential risks– Parallel components
… Gila Basin feasibility study will advise go/no go points for:
1. Development of genetic methods2. Efficacy testing 3. Modeling – to inform components 1, 2 & 54. Target species ecology – to inform 2, 3 & 55. Risk analysis6. Community/public awareness and involvement –
with links to 5, 7 & 87. Seeking regulatory approval8. Post-approval monitoring – to verify 2 & 5
Pro-active Example: Safety First Initiative
2001 – Public workshop obtained extensive feedback on approach2002 – U.S. public-private coalition: Safety First Initiative Executive Advisory Board and Steering Committee 2003 – Kapuscinski et al. Nature Biotechnology 21(6):599-601Propose cross-sectoral working groups to develop safety standards. Partners welcome.
Reports at www.fw.umn.edu/isees
Possible Bureau Roles - Science• Support research and outreach
– inform more pro-active approach– scientific analysis– involve ecologists, conservation geneticists, etc.– representative deliberation
• Provide biosafety research sites– confined field tests– contained labs for fish & other aquatics
• Enhance species and ecological baselines– pre-commercialization studies– post-commercialization monitoring and verification tests– Long-term, large ecosystem scales
‘Coordinated Framework’ for Regulating Biotechnology
• Food and Drug Administration (FDA) claims regulatory lead over transgenic animals, including fish
• Drug regulations forbid public review
• FDA lacks expertise & mandate for F&W
• FWS & NMFS can stop only if harms to threatened or endangered species
Federal Regulation - Uncertainties
• FDA explicitly did not regulate the GloFish:
– “In the absence of a clear risk to the public health, the FDA finds no reason to regulate these particular fish.” (FDA Statement released Dec 9, 2003)
• Where does this leave regulation of environmental safety?
• Authority over biocontrol transgenic animals that are not eaten by humans – such as red shiner, nutria?
Possible Bureau Roles – Resource Management
• Larger role in regulation– biotechnology applied to F&W & natural ecosystems– transgenic fish regulation is a pressing need– Options: from formal MOU with lead agency to
establishing lead authority– Restore transparency of review (NEPA, ESA)
• Establish policies & procedures/standards– GEOs on federal lands– Commenting on other agency actions
• Develop federal GEO monitoring program– tracking spread in the environment– detect unwanted/unexpected problems– safety verification testing
DecisionImplementation
Evaluation
Public Officials
NaturalScientists(few disciplines) Analysis
DefineProblems
SelectOptions
InformationGathering
Synthesis
National Research Council. 1996. Understanding Risk
Public Comment
Dominant Risk Decision Process
Public Demand
Decision
Learning and Feedback
Implementation
Evaluation
AnalysisDeliberation
Public Officials
Natural &SocialScientists
Interested andAffected Parties
AnalysisDeliberation
DefineProblems
SelectOptions
InformationGathering
Synthesis
National Research Council. 1996.
Adaptive management approach “An open process wins every time.” Stu Hann
Adaptive Biosafety Assessment & Management
SetGoals
safe use of GEOs
Information base
Implementationrelease, permits
risk management
Monitoringmark GEOs,databases
ProblemAnalysis
all R & D phases
Policy Designassess risks
identify choices
Kapuscinski et al. 1999
Risk Assessment (or safety verification)
At present, for most transgenic fish: It is very difficult to conduct a reliable lab or confined field test to determine, ahead of time, what is the severity of the environmental harm. However….
As bad asit can get
Verylarge
Sig-nificant
SmallNone
Severity of Harm(Includes Cumulative Effects)
Frequent
Common
Rare
Very closeto never
Very unlikely
Max
imum
Acc
epta
ble
Like
lihoo
d of
Har
m
Safeenough
Not safeenough
It is easier to to determine, ahead of time, the likelihood of environmental harm by a transgenic fish:
• Net fitness methodology
• Integrated confinement system
As bad asit can get
Verylarge
Sig-nificant
SmallNone
Severity of Harm(Includes Cumulative Effects)
Frequent
Common
Rare
Very closeto never
Very unlikely
Max
imum
Acc
epta
ble
Like
lihoo
d of
Har
m
Safeenough
Not safeenough
If the net fitness of the genetically engineered line fits the Purging Scenario.
(If purging in lab test, then purging also likely in more hostile natural environment.)
As bad asit can get
Verylarge
Sig-nificant
SmallNone
Severity of Harm(Includes Cumulative Effects)
Frequent
Common
Rare
Very closeto never
Very unlikely
Max
imum
Acc
epta
ble
Like
lihoo
d of
Har
m
Safeenough
Not safeenough
Stringency of integrated confinement system should reflect predicted risk and severity of harm. Example: high stringency confinement to achieve very low risk if severity is very large.
As bad asit can get
Verylarge
Sig-nificant
SmallNone
Severity of Harm(Includes Cumulative Effects)
Frequent
Common
Rare
Very closeto never
Very unlikely
Max
imum
Acc
epta
ble
Like
lihoo
d of
Har
m
Safeenough
Not safeenough
Hazard scenario determines harms to assess
HAZARD SCENARIO ASSESS ECOLOGICAL CONSEQUENCES
geneflow to
wildrelatives
Net fitness differences between
GEO and wild or feral relatives
Considered safe
Alter genetic
diversity?
Harm species of
special concern?
Reduce community resilience?*
PurgingGEO < wild or feral
assess assess
SpreadGEO ≥ wild or feral
assess assess assess
Trojan geneOpposing traits = population decline
assess assess assess
increasing difficulty
* Resilience could be key question under widespread use of aquatic GEOs
Hazard scenario determines harms to assess
HAZARD SCENARIO ASSESS ECOLOGICAL CONSEQUENCES
Alien species
invasion
Net fitness differences between GEO and wild-type
alien species
Considered safe
Alter genetic
diversity?
Harm species of
special concern?
Reduce community resilience?
DisappearanceGEO < wild-type
assess assess
EstablishmentGEO ≥ wild-type
assess assess
Effective Establishment
Repeated entries
assess assess
increasing difficulty
growth enhanced
Age at maturity
JuvenileViability
Mating Success Fecund.
MaleFertility
AdultViability Scenario
r. trout wild strainDevlin et al. 2001
+? ─amount n/a
+37-83 times
larger
? ? ?candidate
cohoDevlin et al. 1994
+ ? ?early smolt
+ ? ? ? ?for
cohoDevlin et al. 1995
+ ? ?early smolt
+ ? ? ? ? Spread
Nile tilapiaRahman & Maclean 1999
? ? +3 times larger
? ─zero to low
? or
mud loach (huge)Nam et al. 2001
+ ?likely very
early
= ?yolk-sac
absorption
+ ? ? = ? TrojanGene?
medakaMuir & Howard 2001
+12.5% earlier
─30% lower
= +29%
greater
= = Spreadpredicted
Net fitness data missing for most transgenic fishDecreasing influence of trait on net fitness
Pro-active approach example
Involving experts, affected parties, and public at large at key points.
Multi-stakeholder workshop far ahead of possible GE fish introduction in Thailand.
Photos: Mike Morton
NRC 1996. Understanding Risk: Informing Decisions in a Democratic Society
Build higher dikes to resist floods
Multi-layer barriers for effluent from pond drain
Physical confinement - examples
Three Legs of Biotechnology Governance
GovernmentRegulations based
on reliable safety science
Safety professional certification
Producers (businesses & public institutions)
GEO & product safety standardsSafety leadership-top mgm’t to
certified safety professionals
PublicSafety researchSafety education and
trainingSafety deliberation
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