Resource Use Efficiency: Applications of Biotechnology in Genetic

Improvement in Tropical Aquaculture

David J Penman

Institute of AquacultureUniversity of Stirling, Scotland, UK

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

Relevant biotechnologies

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

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

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.

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

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)

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

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

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

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)

Monosex Female Production in XX/XY Species

XX FEMALES

ALL-FEMALE PROGENY (XX)

COMMERCIAL ONGROWING

XX NEOMALES

MIXED SEX FRY (XX,

XY)MTXX NEOMALES, XY MALES

MT

Commercialproduction cycle

MT = 17α-methyltestosterone (or other androgens, depending on species)

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)

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

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

Genomic Location of XX/XY locus in Nile

tilapia in LG1

Palaiokostas et al (2013)

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

Commercialproduction cycle

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)

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

Netherlandso Males need to be killed to obtain milt – problems with

sperm qualityo Infrastructure exists for use of cryopreservation in cattle

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

Use of DNA markers in genetic management of captive populations

Catla catla

hormonal induction of ovulation (above)

Stripping of eggs (right)

Use of DNA markers in genetic management of captive populations

Stripspawning

In vitrofertilization

Singlefamily

Separatetanks 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

SingleTank or hapa

No family i.d.No pedigree data

PIT tagsBiopsy sample

DNAprofiling

Pedigree data

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

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

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

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)

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

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)

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)

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)

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)

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

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

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?