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The Application of Biotechnology in The Application of Biotechnology in LarviLarvi-- and Aquacultureand Aquaculture
YonathanYonathan ZoharZoharCenter of Marine BiotechnologyCenter of Marine Biotechnology
University of Maryland Biotechnology InstituteUniversity of Maryland Biotechnology Institute
Aquaculture: Meeting the ChallengeAquaculture: Meeting the Challenge
•• Currently a 30 million ton, $45 billion industry, Currently a 30 million ton, $45 billion industry, aquaculture must increase production 3aquaculture must increase production 3--4 times by 20254 times by 2025
•• Become more efficient and costBecome more efficient and cost--effectiveeffective•• Overcome biological obstaclesOvercome biological obstacles•• Strong input from modern biology and biotechnologyStrong input from modern biology and biotechnology
Principle Platforms of BiotechnologyPrinciple Platforms of Biotechnology
•• ChromosomeChromosome--set manipulationset manipulation•• FermentationFermentation•• CloningCloning•• Protein expression and productionProtein expression and production•• Gene transfer and Gene transfer and transgenicstransgenics•• Genetic immunizationGenetic immunization•• DNA fingerprints and genetic markersDNA fingerprints and genetic markers•• Genomics Genomics / / bioinformaticsbioinformatics•• Post / functional Post / functional genomicsgenomics
“Bottlenecks” in Commercial “Bottlenecks” in Commercial AquacultureAquaculture
• Reproduction• Early (larval) development • Growth• Nutrition• Disease / health management• Interactions with the environment• “Biosecurity”
Reproduction:Reproduction:The IssuesThe Issues
• No ovulation and spawning• Unpredictable ovulation and spawning• Reproduction / growth interactions• Sterility
Gilthead seabream Striped bass
Captive versus….
Wild fish
Regulation of Fish ReproductionRegulation of Fish ReproductionEnvironmentEnvironment
Brain
GnRHs GnRHs (+)(+) (+/(+/--) ) CatecholaminesCatecholamines
FSHFSH LHLH(+)(+)
Ovulation, spawningOvulation, spawning
Pituitary
Gonads SteroidsSteroidsSteroidsSteroids
SpermiationSpermiation
Fish Fish GonadotropinGonadotropin--Releasing Hormone Releasing Hormone ((GnRHGnRH))
1 2 3 4 5 6 7 8 9 10pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-GlyNH2
Peptide Engineering: Peptide Engineering: GnRHGnRH and and GnRH GnRH AnalogsAnalogs1 2 3 4 5 6 7 8 9 10
pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-GlyNH2
pGlu-His-Trp-Ser-Tyr-DAla-Trp-Leu-Pro-NEt
GnRHa GnRHa and LH Levels After Treatment With Different and LH Levels After Treatment With Different GnRHaGnRHa SustainedSustained--Release Delivery SystemsRelease Delivery Systems
0.01
0.1
1
10
100
0.1
1
10
100
Plas
ma
leve
ls (n
g/m
l)
0.01
2 monthsp[LGA] 7505
3 weeksEVAc
1 monthp[Cpp-sa]
0 35
Plasma levels (ng/m
l)
7 14 21 28 35 0 7 14 21 28 35
0.01
0.1
1
10
100
GnRHa
LH
7 daysp[LGA] 5005100
10
1
0.1
0 7 14 21 287 14 21 28 35
0.010
Time after GnRHa treatment (days)
Application of Application of GnRHaGnRHa Sustained Release Technology to Sustained Release Technology to Induce Ovulation, Spawning and Sperm ProductionInduce Ovulation, Spawning and Sperm Production
•• Gilthead Gilthead seabreamseabream •• Plaice Plaice •• High hatsHigh hats
•• Red Red seabream seabream •• FlounderFlounder
•• Red porgy (Red porgy (PagrusPagrus)) •• SturgeonSturgeon
•• DentexDentex •• Snook Snook
•• European European seabassseabass •• Pompano Pompano
•• Striped bassStriped bass •• Atlantic salmonAtlantic salmon
•• White bassWhite bass •• Coho salmonCoho salmon
•• American shadAmerican shad •• Sockeye salmon Sockeye salmon
•• Gray mulletGray mullet •• Chinook salmonChinook salmon
•• GrouperGrouper •• Rainbow troutRainbow trout
•• Turbot Turbot •• Brown trout Brown trout
GnRHaGnRHa--based delivery systemsbased delivery systems
ImplantsImplants MicrospheresMicrospheres
GnRHGnRH implants are implants are commercially availablecommercially available
Emerging Technologies for the Emerging Technologies for the Manipulation of SpawningManipulation of Spawning
Understand the nature of the captivityUnderstand the nature of the captivity--induced induced alterations in the alterations in the GnRHGnRH system and correct themsystem and correct them
Promoter Reporter: GnRH
InducerInducer (environmental, hormonal)(environmental, hormonal)
GnRH mRNAGnRH mRNA
??
?
?
?FOM, Ovulation & SpawningFOM, Ovulation & Spawning
Early Development:Early Development:The IssuesThe Issues
• Low survival rates• Dependency on live food• Very specific nutritional requirements
“Food Chain” in “Food Chain” in LarvicultureLarviculture
O. O. ZmoraZmora et al., NCMet al., NCM
Heterotrophic Heterotrophic Mass Culture of AlgaeMass Culture of Algae
PhototrophHeterotrophHeterotroph
Dry
Wei
ght (
gr)
ChlorellaChlorella50
40
30
20
10
00 100 200 300 400 500
Time (hours)
Algae Species Grown Heterotrophically
Cyclotella Dunaliella (pigments)Ankistrodesmus EuglenaChlamydomonas NannochloropsisChlorella Nitzschia (EPA)Chlorococcum OchromonasCrypthecodinium (DHA) TetraselmisAmphora Schizochytrium
Lipid and DHA/EPA Ratios in ArtemiaA Comparison of Various Enrichment Diets
Diet Lipid (%) DHA/EPA RatioMicrodiet 15.08 + 1.47 0.78 + 1.47 DHA Selco 20.58 + 0.28 1.4 + 0.1 Algamac 2000 17.70 + 3.80 2.3 + 0.1Aquagrow 26.36 + 2.58 3.75 + 0.17
Harel, Place & Behrens, 2000
Genetic Engineering of Genetic Engineering of Heterotrophic MicroalgeaHeterotrophic Microalgea
Control
lightlight + gludark + glu
Wt Glut1-17
Time (Days)
Zaslavskaia et al., Science, June 2001Glut1-GFP transformed
Delivery of Beneficial Compounds to Larvae Delivery of Beneficial Compounds to Larvae (The Ghent Group!)(The Ghent Group!)
•• ImprovedImproved•• Genetically modifiedGenetically modified
•• NutrientsNutrients•• TherapeuticsTherapeutics•• VaccinesVaccines•• HormonesHormones•• EnzymesEnzymes•• PigmentsPigments•• DNADNA
RecombinantRecombinant(algae, bacteria, (algae, bacteria,
yeast)yeast)
BC,MDBC,MD
Fatty acid concentration in enriched rotifers
ratio DHA/EPA1.6
0
10
20
30
40
50
60
70
80
90
100
110
CS nanno Super DHA DHA8
DHAEPA
0.7 0.3 7.8mg/
g dr
y w
eigh
t
P. Sorgeloos, Ghent University
Recombinant Growth Hormone: Enhancing Larval Recombinant Growth Hormone: Enhancing Larval Growth and Survival in Growth and Survival in SeabreamSeabream
BenBen--AtiaAtia et al., 1999et al., 1999
Growth:Growth:The IssueThe Issue
• Long time to market size
“Extraordinary Salmon Growth”“Extraordinary Salmon Growth”ssGHssGH--I genomic DNAI genomic DNA
ATGATG TAGTAGssMTssMT promoterpromoter
Devlin et al., 1994(coho salmon)
Growth Rate in Transgenic Atlantic SalmonGrowth Rate in Transgenic Atlantic Salmon
May Jun Jul Aug Sept Oct
100g
200g
300g
400g
500gTransgenics
Control
Wei
ght (
gr.)
Hew, C.L. and Fletcher, G., 1997
Transgenic, Transgenic, Fast Growing Fast Growing Salmon Are Salmon Are
Commercially Commercially AvailableAvailable
Transgenic Fish: Public ConcernsTransgenic Fish: Public Concerns
Time Magazine,March ‘00
Transgenic Fish:Transgenic Fish:Addressing Public ConcernsAddressing Public Concerns
•• Use “all fish” constructs• Avoid viral promoters • Make the transgenic fish sterile• Contain GM fish in recirculated aquaculture
systems
Enhancing Muscle Growth by Regulating Enhancing Muscle Growth by Regulating MyostatinMyostatin (GF) Activity(GF) Activity
Lee and McPherron; PNAS, August 2001
Disease / Health Management:Disease / Health Management:The IssuesThe Issues
• Intensification of aquaculture is accompanied by increased susceptibility to pathogens
• Losses due to disease are in the billion $ range annually
Animal Health: Animal Health: The ChallengesThe Challenges
•• Early diagnostics (histology, immune, molecular)
• Efficient vaccination (microbes, expressed antigens, DNA)
“Real-time” Quantitative RT-PCR: TaqMan
Science (1999) 284 (5413): 449-450
ThresholdCt
Genetic / DNA VaccinationGenetic / DNA Vaccination
Pathogen
genetic material
gene for antigen plasmid
altered plasmid
syringe ⇒ muscleplasmids
gene gun ⇒ skinparticles
Genetic Immunization Against Infectious Genetic Immunization Against Infectious Hematopoietic Hematopoietic Necrosis (IHN) VirusNecrosis (IHN) Virus
GlycoproteinGlycoprotein
Days postDays post--challengechallenge%
Cum
ulat
ive
mor
talit
y%
Cum
ulat
ive
mor
talit
yIHN VirusIHN Virus
pCDNA pCDNA 3.13.1
Splice/Poly A
Amp.Amp.
CMVCMVPromoterPromoter
G geneG gene
ORIORI Anderson, Mourich, Fahrenkrug, LaPatra, Shepherd & Leong, 1996
Fish Vaccination Fish Vaccination -- Labor IntensiveLabor Intensive
Interactions with the Environment:Interactions with the Environment:The IssuesThe Issues
Naylor et al.; Science, 2000 Naylor et al.; Science, 2000
Interactions with the Environment:Interactions with the Environment:The IssuesThe Issues
• Effect of aquaculture on the environment- Chemical pollution (nutrients, antibiotics) - Biological pollution (escapees, GMOs, disease)
• Effect of the environment on aquaculture- Pollutants- Algal blooms- Pathogens- Suboptimal conditions
RecirculatedRecirculated, Fully Contained , Fully Contained Marine AquacultureMarine Aquaculture
• No pollution, disease-free, clean, flexible, generic,GMOs and non-native species
• Applicable for rural and urban locations
Characterization and Improvement of Characterization and Improvement of Microbial Communities in Microbial Communities in BiofiltersBiofilters
16S 16S rRNA rRNA analysisanalysis
DGGE fingerprintingDGGE fingerprinting
Gene Transfer, Gene Transfer, TransgenicsTransgenics::Potential ApplicationsPotential Applications
•• Improve performances Improve performances Reproduction; early development; growth; digestion; Reproduction; early development; growth; digestion; disease resistance; aggressiondisease resistance; aggression
•• Develop new feeds Develop new feeds PlantPlant--stuff with adequate proteins and lipidsstuff with adequate proteins and lipids
•• Tailor fish to the marketTailor fish to the marketTaste; flesh color; fat content; body formTaste; flesh color; fat content; body form
•• Basic researchBasic researchReproduction, development, growth, immunologyReproduction, development, growth, immunology
Post / Functional Post / Functional GenomicsGenomics::From Genes to FunctionsFrom Genes to Functions
• DNA microarrays / gene chips
Measure differential gene expression of the entire genome on 1 chip
• PROTEins encoded by the genOMEFull array of all proteins present in an organism, a tissue or a cell
• Protein microarrays / chipsMeasure differential abundance of thousands of proteins on one chip (with specific antibodies)
• Tissue microarrays / chipsMonitor gene and protein expression patterns in hundreds of tissues on one chip
• Protein activity chip…. (still a dream…)
DNA DNA Microarrays Microarrays / Gene Chips/ Gene Chips
Fast growth; healthyFast growth; healthy Slow growth; diseasedSlow growth; diseased
mRNAmRNA mRNAmRNA
cDNAcDNA cDNAcDNA
Functional Genomics: Gene Chips
Robotic ‘arrayer’
The entire yeast genome on a chip!; Dr. Patrick Brown, Stanford U.
Acknowledgements:Acknowledgements:
• Drs. J. Trant, J. Du, A. Place, G. Vasta, M. Harel, H. Schreier, V. Frenkel- COMB, Baltimore, MD
• Dr. Amos Tandler, Odi Zmora, NCM, Eilat• Dr. Patrick Sorgeloos, University of Ghent• Dr. Choy Hew- National U. of Singapore• Dr. Scott Lapatra- Clear Springs Foods Inc., Idaho• Dr. Paul Behrens, Martek Biosciences, MD
Microinjecting DNA into Microinjecting DNA into zebrafishzebrafish embryosembryos
Proteome Proteome AnalysisAnalysis
egg proteinsegg proteins egg proteinsegg proteins
QuickTime™ and aPNG decompressor
are needed to see this picture.
identificationcharacterization
2D PAGE analysis
Genetic Selection: Genetic Selection: Integrating Molecular GeneticsIntegrating Molecular Genetics
• Identify beneficial traits:Growth rate; food conversion; flesh quality; fecundity; age at maturity; disease resistance
• Identify and map the relevant gene(s):Single genes; multiple genes (QTLs)(Danzmann et al., 1999; Rodriguez et al., 1999; Davis and Hetzel, 2000)
• Link gene markers to gene(s) of interest• Perform marker-assisted selection
Gene Linkage Mapping in Gene Linkage Mapping in Crustaceans and FinfishCrustaceans and Finfish
• Zebrafish Kelly et al., 2000• Salmon Lie et al., 1997• Catfish Liu, 1999• Tilapia Kocher et al., 1999• Kuruma shrimp Moore et al., 1999• Tiger shrimp Moore et al., 1999
Gene Transfer, Gene Transfer, TransgenicsTransgenics::Future DirectionsFuture Directions
•• Better delivery of Better delivery of transgenes transgenes Vectors; sperm; Vectors; sperm; lipofectionlipofection; gene guns ; gene guns
•• Better integration of Better integration of transgene transgene Transposable elements (Transposable elements (IvicsIvics, Z., , Z., IzsvakIzsvak, Z. & Hackett, P.B., , Z. & Hackett, P.B., 1999); NLS (1999); NLS (CollasCollas, P., , P., HusebyeHusebye, H. & , H. & AlestromAlestrom, P., 1996), P., 1996)
•• Knock out /knock in technologyKnock out /knock in technologyEmbryonic stem cells (Embryonic stem cells (Hong, S., Chen, S. & Hong, S., Chen, S. & SchartlSchartl, M., 2000), M., 2000)
•• Spatial and temporalSpatial and temporal--specific expression specific expression Promoters; constructsPromoters; constructs
Expression of Recombinant Substances:Expression of Recombinant Substances:Potential ApplicationsPotential Applications
•• Hormones (GH, Hormones (GH, ProlProl, , GtHsGtHs, , GFsGFs,…),…)•• Protective antigens (viral coat proteins,...)Protective antigens (viral coat proteins,...)•• Enhancers of immune system (Enhancers of immune system (glucansglucans,...),...)•• Antibacterial compounds (peptides, Antibacterial compounds (peptides, lysozymeslysozymes))•• Enzymes (Enzymes (phytasephytase, , cellulasecellulase, , desaturasedesaturase,...),...)•• Others (antifreeze proteins,…) Others (antifreeze proteins,…)
Regulation of Fish ReproductionRegulation of Fish ReproductionEnvironmentEnvironment
Brain
GnRHs GnRHs (+)(+) (+/(+/--) ) CatecholaminesCatecholamines
FSHFSH LHLH(+)(+)
Ovulation, spawningOvulation, spawning
Pituitary
Gonads SteroidsSteroidsSteroidsSteroids
SpermiationSpermiation
Genetic Engineering for the Genetic Engineering for the Control of ReproductionControl of Reproduction
InduceInducerr (water, feed)(water, feed)
Inducible Promoter Anti-GnRH or GnRH
AntiAnti--GnRH mRNAGnRH mRNA GnRHGnRH
SterilitySterility SpawningSpawning
Uzbekova, Alestrom, Hanley & Breton, 2000
Peptide Engineering: Peptide Engineering: GnRHGnRH and and GnRH GnRH AnalogsAnalogs1 2 3 4 5 6 7 8 9 10
pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-GlyNH2
pGlu-His-Trp-Ser-Tyr-DAla-Trp-Leu-Pro-NEt
Ultrasound Enhanced Uptake of Ultrasound Enhanced Uptake of CalceinCalcein
w/o ultrasound w/ ultrasound0
30
60
90
120
150
aver
age
pixe
l lum
inos
ity (0
- 25
5)
n = 5
n = 5
w/o ultrasound w/ ultrasound
aver
age
pixe
l lum
inos
ityCalcein w/o ultrasound
Calcein w/ 30 second ultrasound
Frenkel et al., 2000
UltrasoundUltrasound--mediated delivery of mediated delivery of biopolymer/DNA biopolymer/DNA nanospheresnanospheres
over
head
≈ 10 µm
cros
s sec
tions
control
skin
su
rfac
e
US (60 s)⇓
particles
particles US (40 s)⇓
particlesFrenkel et al., 2000
Biological FiltrationBiological Filtration
NitrosomonasNitrosomonas NitrobacterNitrobacter
NHNH33 NONO22-- NONO33--AmmoniaAmmonia NitriteNitrite NitrateNitrate
SeaweedSeaweed
AnaerobicAnaerobic
NN22
Injection-20% spawning
Sustained release90% spawning
Plasma hormone levels and ovulation in striped bass females Plasma hormone levels and ovulation in striped bass females after treatment with after treatment with GnRHaGnRHa--delivery systemsdelivery systems
GnRHaGnRHa--implantimplantGnRHaGnRHa--microspheresmicrospheres
88
1100
00
2200
4400
77 1144 2211
Time after Time after GnRHaGnRHa treatment (days) treatment (days)
Plas
ma
horm
one
Plas
ma
horm
one
ngng// m
Lm
L
00 77 1144 2211 2200..0011
00..11
11
1100
Cum
ulative ovulation (%)
Cum
ulative ovulation (%)
LHLHGnRHaGnRHa110000 110000
880088
660066
44
22
00
0000 77 1144 2211 2288
Monitoring Larval Development Through Monitoring Larval Development Through Gene Expression: Real Time RTGene Expression: Real Time RT--PCRPCR
TaqmanTaqman
ThresholdCt
GeneGene--specific specific primerprimer
GeneGene--specific primerspecific primer
GeneGene--specific specific probe with probe with fluorfluorand quencherand quencher
FluorFluor dissociates dissociates from quencherfrom quencher
Nutrition:Nutrition:The IssuesThe Issues
• Food cost accounts for 50% of growout expenses• Aquaculture industry spends $ 6 billion/year on
feed• Projections for 2010 are $ 10 billion• Heavy dependence on fish meal (1/3 of all
fisheries; 6 mmt/year)
Source: USDA; FAO
The Immediate Challenge: The Immediate Challenge: Replace Fish Meal with Plant MealReplace Fish Meal with Plant Meal
• “Production of fatty acid components of high value drying oils in transgenic soybean embryos”Cahoon et al.; PNAS, 1999
• Develop “algal meal”
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