Resource use efficiency in fish: Application of biotechnology in genetic improvement in tropical aquaculture presentation by David Penman, University of Stirling, Stirling, United Kingdom

1. Resource Use Efficiency: Applications
of Biotechnology in Genetic
Improvement in Tropical Aquaculture
David J Penman
Institute of Aquaculture
University of Stirling, Scotland, UK

2. Scope of talk
• This talk will cover biotechnologies (as understood
from prior FAO definitions) related to genetic
improvement in tropical aquaculture
• It will attempt to look at these in the context of
(improving) resource use efficiency
• Focus on (fin)fish species
• Globally, aquaculture ranges from well-established
domesticated species to capture and ongrowing of
wild organisms – this talk will try to reflect this

3. Relevant biotechnologies
• Chromosome set manipulation
• Sex ratio manipulation
• Cryopreservation
• DNA markers, linkage mapping, QTLs, etc
• GM technologies

4. Relationship with selective breeding
• Selective breeding is not included in the scope of
this talk (not considered as a biotechnology)
• However, many of the relevant biotechnologies
are used in the context of managing captive
breeding programmes and genetic improvement
by selective breeding
• So, several aspects of the talk will require
reference to breeding programmes in relevant
species

5. Biotechnologies in Global Aquaculture
• Most of the relevant biotechnologies that I will
describe have been applied to a greater extent in
non-tropical aquaculture, particularly well-
established, high-value species such as salmonids
• I will this draw on some examples from such
species/culture systems, to illustrate the current
trends and the directions that may be followed in
tropical aquaculture.

6. The context of applying
biotechnologies in aquaculture
• Use of chromosome set and sex ratio manipulation in
controlling maturation and reproduction
• Use of cryopreservation in gene banking, transfer of
genetic material and assessing genetic gain
• Use of DNA markers in understanding population
structure of wild genetic resources
• Use of DNA markers in genetic management (Ne,
inbreeding) of captive populations
• Use of DNA markers/genomics as tools in enhancing
selective breeding
• Use of GM and related technologies in enhancing
performance in aquaculture

7. Triploidy
• Widely used in rainbow trout, Pacific oyster,
Atlantic salmon in temperate aquaculture to
control maturation/reproduction
• Needs unfertilised eggs and sperm to allow
pressure or temperature shocking of newly
fertilised eggs
• Has been tested in Nile tilapia, including field
trials in Africa, with very promising results but
incompatible with breeding systems in
commercial hatcheries (many females, produce
small batches of eggs frequently and
asynchronously: embryos or fry collected later)
• Potential in other species (PTO)

8. Grass carp – biological containment
Although no requirement for triploidy in grass carp aquaculture
in major producing countries, triploidy is widely used in the
southern USA where grass carp is an exotic species used to
control aquatic plant growth (diploids banned in some states)
Juvenile grass carp Screening blood samples to test triploidy

9. Alternative method of control of exotic
species (silver and bighead carp in USA)!

10. African catfish
(Clarias gariepinus)
• Culture is booming, particularly in Nigeria, with
interesting peri-urban production systems growing
• Only one formal breeding programme (WFC, Egypt) –
this aspect needs development in SS Africa to
support sustainable growth
• Genomics/genetics resources being developed
(Hungary/UK/Nigeria/Netherlands)
• Where market size is large (> 1 kg), ovarian
development in females can be significant (20% of
total weight): triploidy could eliminate this

11. Control of sex ratio
• Desirable in species where one sex grows more
slowly and/or matures earlier than the other, or
where both sexes mature and breed before
harvest
• Sex determination in fish is very varied –
sometimes XX/XY or WZ/ZZ, can be polygenic or
influenced by environment (temperature during
differentiation), also find hermaphroditic species
(e.g. grouper, barramundi)

12. Monosex Female Production in XX/XY Species
XX FEMALES
ALL-FEMALE PROGENY (XX)
COMMERCIAL ONGROWING
XX NEOMALES
MIXED SEX
FRY (XX, XY)
MT
XX NEOMALES, XY MALES
MT
Commercial
production cycle
MT = 17α-methyltestosterone (or other androgens, depending on species)

13. Monosex female production in the
silver barb (Barbonymus gonionotus)
• Female grows faster than male, ovaries also eaten
• Technique for monosex female production developed
in Thailand in 1990s, used in aquaculture but not
now used due to decline in popularity of species in
aquaculture
(snipview.com)

14. Mixed Sex v’s Monosex Tilapia
(photo by GC Mair)
MST SRT/GMT ®
MST = mixed sex tilapia; SRT = sex-reversed tilapia; GMT = genetically male tilapia

15. Control of Maturation/Reproduction in Nile
Tilapia (Oreochromis niloticus)
Hormonal masculinization (MT in-feed)
• Can be very effective (but often not very well done!), most
commonly used technique, banned in several major countries
(not always enforced)
GMT (YY males x XX females -> XY males)
• Has been used on a small scale commercially, hindered by
complexities of sex determination: XX/XY locus, but other
genes and temperature [in some families] can affect sex
determination, YY production process complex.
• Genomic analysis being used to develop sex-linked markers
and marker-assisted selection to improve GMT

16. Genomic Location of
XX/XY locus in Nile
tilapia in LG1
Palaiokostas et al (2013)

17. Monosex Male Production in WZ/ZZ Species
ZZ MALES
“ALL-MALE” PROGENY (ZZ)
COMMERCIAL ONGROWING
ZZ NEOFEMALES
MIXED SEX
FRY (WZ, ZZ)
WZ FEMALES, ZZ NEOFEMALES
Commercial
production cycle

18. Genetic sex control in a WZ/ZZ species – the
giant freshwater prawn
• Males grow faster than females in the giant
freshwater prawn Macrobrachium rosenbergii.
• Feminization of ZZ males achieved by surgical
removal of androgenic gland.
• Developed by Amir Sagi’s group: mass
production 2006, used on small scale in
Thailand, India, Vietnam…
• More recently developed RNAi technique
(Aflafo et al 2015) for feminization: double-
stranded RNA injection caused temporary
silencing of expression of insulin-like androgenic
gland hormone (dsRNA degraded rapidly)
• Argue that RNAi is a safe biotechnology for this
and other uses in aquaculture
ZZ neofemale prawn (above) and
harvest of all-males (below)
(U. Na-nakorn)

19. Cryopreservation
• Not widely used in commercial aquaculture
• Useful for gene banking of founder populations,
efficient for assessing genetic gain (cryo x current gen
v’s current x current)
• Interesting case study in Nigeria:
o Cryopreserved milt used to transfer genetic material from
Netherlands
o Males need to be killed to obtain milt – problems with
sperm quality
o Infrastructure exists for use of cryopreservation in cattle

20. Use of DNA markers in genetic
management of captive populations
• Many species of fish can be stripped of eggs and
sperm manually, then individual families can be set
up and maintained
• For some, this is feasible experimentally but not on a
commercial scale
• For others, mass spawning is still the only feasible
way of producing fry
• The way fish are bred has consequences for
establishing pedigree and controlling Ne/inbreeding

21. Use of DNA markers in genetic
management of captive populations
Catla catla
hormonal induction of ovulation (above)
Stripping of eggs (right)

22. Use of DNA markers in genetic
management of captive populations
Strip
spawning
In vitro
fertilization
Single
family
Separate
tanks or hapas
PIT
tags
Pedigree
data
 Stripping and in vitro fertilization make control over pedigree feasible:
 Mass spawning makes this impossible without the use of DNA markers:
Mass spawning
and fertilization
Mixed
families
Single
Tank or hapa
No family i.d.
No pedigree data
PIT tags
Biopsy sample
DNA
profiling
Pedigree
data

23. Consequences
• For many highly fecund species, eggs are small and
survival rates very variable, generally low
• No control over family contribution to next
generation of broodstock -> uneven contribution,
lower Ne, inbreeding starts
• Control allows more even contribution, higher Ne,
also basis for sustainable selective breeding
programmes

24. Example - Milkfish (Chanos chanos)
• Important aquaculture species in SE Asia: > 1 million t p.a.
• Catadromous, high fecundity, small eggs, long generation time (5-7
years), large broodstock – high investment in broodstock
• DNA microsatellite markers developed for parental allocation to
allow control of family size/Ne in captive-reared broodstock
• Sex-linked markers (not developed yet) would aid in ensuring both
males and females retained in all families

25. Use of DNA markers/genomics as tools
in enhancing selective breeding

26. Genetic Improvement of Farmed Tilapia (GIFT)
• The first major breeding programme for a tropical
aquaculture species – Nile tilapia
• Started 1988, now >20 generations
Rearing of separate families
in hapas (GIFT Manual,
WorldFish Center)

27. Genetic Improvement of Farmed Tilapia (GIFT)
• Multinational, public funding, dissemination to many
countries
• Core breeding programme run by WorldFish Centre
(Penang)
• Pedigree established using single pair matings,
separate family rearing, PIT tags (no DNA markers)
• First private offshoot uses DNA markers, claims faster
progress (hard to verify – little information)
• Several other secondary breeding programmes in a
range of countries, plus other tilapia breeding
programmes

28. DNA markers for parental allocation in
common carp (Vietnam)
• Earlier mass selection programme showed gain for five
generations then stopped (inbreeding/loss of genetic variation)
• More recent family-based programme: (i) separate or (ii)
communal rearing with parental assignment using DNA markers?
• Tested in parallel – same families, split
Ninh et al. (2011)
Ninh et al. (2013)

29. DNA markers for parental allocation in
common carp (Vietnam)
• Heritabilities moderate to high in both
environments
• Reduced maternal and common environmental
effects under communal rearing
• Fish grew faster under communal rearing –
reduced generation time
• Greater response to selection under communal
rearing
• Perhaps surprisingly, communal rearing and use
of DNA markers was cheaper than separate
rearing (hapas, extra labour costs)

30. QTLs and MAS
• Marker-assisted selection (MAS) for quantitative trait loci (QTL)
affecting complex traits such as disease resistance offers more
efficiency than phenotypic selection
• MAS for Infectious Pancreatic Necrosis (viral) in Atlantic salmon, in
both Scotland and Norway, was the first example of such an
application in a commercial breeding programme in aquaculture.
QTL in LG21
• Patent has been applied for based on this (WO 2014006428 A1)
Houston et al. (2010)

31. QTLs and MAS
• Many other QTL have been mapped in aquaculture species
• E.g. pearl traits in pearl oyster Pinctada maxima (also GWAS)
• Slow uptake so far, expect to see many more over next few
years
• Also now seeing many SNP chips being developed (including
for tilapia), application of these in breeding programmes
being developed
• Also seeing breeding companies developing use of genome-
wide selection (still in early stages in aquaculture)

32. Very long-running evaluation for food safety in USA
FDA issued “Preliminary Finding of No Significant Impact” (May 2012)
http://www.fda.gov/AnimalVeterinary/DevelopmentApprovalProcess/GeneticEngineering/GeneticallyEngineeredAnimals/ucm280853.htm
AquaBounty cleared to produce salmon eggs in Canada for commercial
purposes (Nov 2013)
http://aquabounty.com/wp-content/uploads/2014/02/2013-11.25-AquAdvantage-Salmon-Cleared-for-Production.pdf
Aquabounty fined by Panama for regulatory failures in its Panama plant
(The Guardian, 29/10/14)
If successful in coming to market, this could be a watershed for the
development of GM animals – many other developments in the pipeline
GM Atlantic Salmon

33. Transgenics v’s “gene editing”
• Sledgehammer v’s scalpel?
• Range of techniques (e.g. CRISPR/Cas9)
• Can be used to modify/knockdown genes or alleles of genes
• Could be used to affect a wide variety of traits in a cost-
effective fashion, including disease resistance, sexual
development, …..
• Should this be treated in the same way as transgenic organisms
(poorly targeted introduction of modified genes, often from
other organisms)?
Li et al. (2014)
Germ cell
development
manipulation in
Nile tilapia via
Nanos genes

34. Summary
• Aquaculture covers species from well-deloped
breeding programmes to wild seed
• A range of biotechnologies are being applied in
commercial aquaculture
• Most advanced in temperate/high-value species
• Relatively limited application in many tropical
aquaculture species to date
• Genomics and NGS are contributing to the scope and
pace of development of new biotechnologies and
application to aquaculture
• Bright future?