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.

0

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Per

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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 resistance

Virus 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) #

Bioremediation

Mercuric ion reductase (from E. coli), cytochrome P450 (from human)

poplar#

Pharmaceuticals, biologics, industrial chemicals

swine viral vaccine, avidin, trypsin, high laurate many others in research

corn - swine vaccine #, avidin**, trypsin *,

canola - laurate *numerous other crops

Rehabilitate endangered species

Fungal 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 Assessment

1. 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 Management

Risk 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

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0.1000

0.1250

400 (a)

400 (b)

400 (c)

67 (a)

67 (b)

67 (c)

treatment (sGH line number)

tran

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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 hybridization

2. 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-scale

Scientists’ 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 Harm

Density-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 li

kel

ihoo

d (

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 Harm

Low High

NRC 2004. Miller et al. 2002

Ris

k (L

ikel

ihoo

d of

Har

m)

Frequent

Very closeto never

Severity of Harm

NoneAs 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

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Severity of Harm(Includes Cumulative Effects)

Frequent

Common

Rare

Very closeto never

Very unlikely

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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

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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 methods

2. Efficacy testing

3. Modeling – to inform components 1, 2 & 5

4. Target species ecology – to inform 2, 3 & 5

5. Risk analysis

6. Community/public awareness and involvement –with links to 5, 7 & 8

7. Seeking regulatory approval

8. 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

Decision

Implementation

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

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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

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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

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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

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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

Juvenile

Viability

Mating

Success Fecund.

Male

Fertility

Adult

Viability 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

+ ? ? = ? Trojan

Gene?

medakaMuir & Howard 2001

+12.5% earlier

─30% lower

= +29%

greater

= = Spread

predicted

Net fitness data missing for most transgenic fish

Decreasing 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

Recirculating Aquaculture Systems

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