Considerations for Genetic

Improvement in Penaeid Shrimp

C. Greg Lutz
Traditional Animal Breeding
Approaches

Additive Genetic Effects:

Selection

Dominance Genetic Effects:

Crossbreeding
Hybridization
Inbreeding

Relationships, correlations:
growth, reproduction,
resistance
 Strain Effects: The Population Genetic
Structure of Pacific White
Original Selection: Shrimp (Litopenaeus
vannamei) from Mexico
Natural Selection to Panama: Microsatellite
DNA Variation
Valles-Jimenez, R; Cruz, P;
Perez-Enriquez, R. Marine
Biotechnology6. 5 (Oct
2004)
 Additive Genetic Effects:
ARTIFICIAL SELECTION

SELECTION: the animal on top will be better breeding stock

IF: growth is the goal AND IF: growth is moderately heritable in
this population. AND IF: these animals were raised together
Additive Genetic Effects:
SELECTION How does it work?

Additive: Big x Big = Larger

But only partially?
Transmitted across generations
 The variation that we can observe includes various components:
Genetic variation, variation caused by the environment, and
random variation resulting from the circumstances experienced by
each individual
Heritability
h2 = VA/VP

PADRES

CRIAS
Preston et al. 2004. Comparative
growth of selected and non-
selected Kuruma shrimp Penaeus
(Marsupenaeus) japonicus in
commercial farm ponds;
implications for broodstock
production. Aquaculture 231:73-
82.
 Additive Genetic Effects: Heritability of GROWTH
Characteristic h2 : Heritability Study

Total Length 0.22 + 0.07 Perez-Rostro and Ibarra, 2003 - Mexico

0.39 + 0.03 Andriantahina et al., 2012 - China
Total Weight 0.17 + 0.06 Perez-Rostro and Ibarra, 2003 - Mexico
0.24 + 0.05 Gitterle et al., 2005 - Colombia
0.17 + 0.04 Gitterle et al., 2005 - Colombia
0.24 to 0.35 vs 0.37 to 0.45* Castillo-Juarez et al., 2007 Mexico
0.21 + 0.04 Campos-Montes et al., 2013 - Mexico
0.52 + 0.03 Andriantahina et al., 2012 - China
0.19 + 0.03 Caballero-Zamora et al., 2015 - Mexico

{ 0.15 to 0.33 no WSSV

0.09 to 0.11 with WSSV
Caballero-Zamora et al., 2015 Mexico

In real-world trials, selection gains for growth typically range from 2% to 21% per
generation
AND not just for growth h2s for
Arcos, Racotta and
Ibarra, 2004 Mexico,
Hatchery and
vannamei Reproductive
Heritabilities: h2 Traits
0.54 for days to
spawning following
ablation
0.17 for number of
eggs produced
Ibarra et al., 2005
Mexico, vannamei
Heritability of 0.20 for
total number of
spawns
Question: How would this
relate to inbreeding within a
closed hatchery population?
Vela-Avita et al. 2013. Decline of
genetic variability in a captive
population of Pacific white shrimp
Penaeus (Litopenaeus) vannamei
using microsatellite and pedigree
information. Electronic Journal of
Biotechnology ISSN: 0717-3458
Vol. 16 No. 4.
Domestication/Selection Reduces Genetic
Variation
an inconvenient reality when you only
allow a select portion of the population to
reproduce. However, often with negligible
effects.

Comparisons between a wild population

collected from the Mexican Pacific
Ocean and two different generations (7
and 9) from a captive population
selected for growth and survival. Using
26 loci, both captive populations
showed a decline in the expected
heterozygosity (20%) and allelic
diversity indices (48 to 91%) compared
to the wild population (P < 0.05).
Effective population size estimates
declined from 48.2% to 64.0% (P < 0.05)
compared to the wild population.
Approaches to Selection
Mass Selection: Should normally be used unless there are
significant reasons for choosing other approaches.

Family Selection: Preferable when the trait being improved

has a LOW heritability. Families should be raised together.
Works better (but not necessarily preferable) when family
size is large.

Within-Family Selection: Usually a way to deal with

environmental influences common to members of a family.

In all of these approaches, the influences of h2, family size,

c2 (common influences like early environment), and mating
system have major impacts.
Common environment effects!
C2
Positive or Negative covariance
within a family group.
Can cause problems in Family
Selection, and in Mass Selection.
Sometimes
the unit we
select on IS
the family

So selection is based
on using additive
genetic effects, which
are transmitted directly
from an individual to its
offspring.
Dominance Genetic Effects:
Used in Crossbreeding and
Hybridization. NOT transmitted
directly dependent on
combinations of parents.
Dominance
Genetic Effects:
Crossbreeding Within a
Species
Goyard et al., 2008 New Caledonia
In stylirostris, crossbreeding distinctly different
strains:
Improved growth rate by 37% (+ 7%)
Improved survival
Improved production by 40% in year 1
and 130% in year 2

Yao et al., 2007 China

In vannamei, crossbreeding different strains:
Improved growth rate and survival simultaneously

Why dont more hatcheries do this?!!! Its not difficult. I suspect its because:
You dont need a consultant to do this, so THEY dont recommend it
 Dominance Effects in the Real World including Reciprocal Effects

$4.500,00

$4.000,00

$3.500,00

$3.000,00

$2.500,00
$/ha por Origen

$2.000,00
Dominance : has a dark side INBREEDING!!!
Moss et al., 2008 - Hawaii
Mated full siblings in successive
generations. Depresion por Endogamia.
For every 10% increase in inbreeding:
Hatch rate decreased by 12.3%
Hatchery survival decreased by 11.0%
Growout survival decreased by 3.8%

De Donato et al., 2005 Venezuela

Over 11 generations in a closed population (no new blood
lines):
Survival INCREASED from 59% to 76%
Growth INCREASED from 0.76 g/wk to 0.87 g/wk
FCR DECREASED from 1.86 to 1.51 kg feed/kg harvest
Production INCREASED from 1.2 to 2.1 MT/ha
Deformities DECREASED from 29% to 1%
Should We Fear Inbreeding?
The Santa Gertrudis Breed An
entire breed established from ONE
(1) bull named Monkey

Now found throughout the world.

A valuable and adaptable breed.
Pastor
Aleman
 Y Pastor
que??
Aleman
Se
burlan
de mi
Each locus (point on a pair of chromosomes that
codes for a specific product) has two alleles
(forms) one from each parent. Inbreeding
Coefficient is the probability that both the alleles a A
at a locus are identical BY INHERITANCE.

Remember:
Inbreeding /
Inbreeding
Depression

In Fitness-Related Traits
mm Mm MM adequacion (growth, fecundity,
resistance): Directional Dominance
INBREEDING

mm MM
X
Heterozygotes (Mm) are more similar
to individuals that are homozygous for
the dominant allele (MM).

Inbreeding reduces the number of

heterozygotes, and the overall average
fitness is reduced.

NOTE: Gene frequencies have not

changed.
 In Fitness-Related Traits:
The more similar the Directional Dominance
heterozygous animals (Hh) are to
those that are homozygous for
the dominant allele (HH), the
greater the reduction in fitness
as inbreeding accumulates.

hh Hh HH

INBREEDING

hh HH
X
De Donato et al. 2005. Mass
selection and inbreeding effects on
a cultivated strain of Penaeus
(Litopenaeus) vannamei in
Venezuela. Aquaculture 247:159-
167.
Founder Population of only
~100 animals most from
Mexico, and already somewhat
inbred. Once established, 1500
broodstock selected from 150 ha 350 ha 438 ha

15000 animals (10%) each year.

The consultant will tell you a
family selection program must be
established or you will go out of
business in 2 years.
74,460,000 larvae stocked
per cycle from 1997 onward.
Roughly 100,000 per female.

150 ha 350 ha 438 ha

But what happens when SEVERE

inbreeding occurs??? Lets see!
Keys et al. 2004. Comparative
growth and survival of inbred and
outbred Penaeus (Marsupenaeus)
japonicus, reared under controlled
environment conditions:
indications of inbreeding
depression. Aquaculture 247:159-
167.

In this EXQUISITE study

levels of inbreeding of F=
2831% had consistently
negative yet insignificant
affects on growth, survival
and yield of domesticated P.
japonicus, suggesting that
inbreeding levels below
F=31% may be tolerated by
penaieds although the
negative trends may
continue at higher levels.

We will put this in context

de los Rios-Perez et al. 2015.
Inbreeding effects on body
weight at harvest size and grow-
out survival rate in a genetic
selected population of Pacific
White shrimp Penaeus
(Litopenaeus) vannamei.
Journal of the World
Aquaculture Society 46(1):53-60.

Starting with a line already

selected for growth and
survival, 320 families were
produced with inbreeding
coefficients ranging from 0 to
60.4%. For every 10% increase
in inbreeding, body weight
decreased by 2.19% + 0.41%.
However, survival was
unchanged (-0.009% + 0.006%).

Why no impact on survival?...

 Dead shrimp is still dead shrimp, even if
Yang et al. 2007. Effect of death comes 14.6 hr later.
inbreeding on growth and WSSV
resistance of the juvenile Chinese
shrimp, Fenneropenaeus chinensis.
Shuichan Xuebao 31.2:226-234

Compared to wild shrimp, an

inbreeding coefficient of 25% in a
captive population reduced
average weight from 1.58 + 0.01 g
to 1.43 + 0.04 g (about 10%
overall, or roughly 4% per every
10% increase in F).

Similarly, average time to

death after infection with
WSSV was reduced from
100.43 + 0.68 h to 85.84 +
1.70 h. OMG!!!!
 Per 10% increase in inbreeding
Survival (history
Growth is important) Source
P. (L.) vannamei Increased + Increased + De Donato et al. 2005
-2.6% to -3.9% -0.1% to 0.5% N.S. Moss et al. 2007
- -3.80% Moss et al. 2008
-2.14% -0.01% N.S. de los Rios-Perez et al. 2015
F. chinensis -3.06% to -4.74% -0.85% to -2.49% N.S. Luo et al. 2014

EL CANDADO: Normal:
Survival
COPY PLs with 13.00 g at (history is
Normal:
0.25 inbreeding Growth harvest important) 74%
P. (L.) vannamei
Moss et al. 2007 -6.5% to -9.75% 11.73g to 12.16g -0.25% to 0.5% 73.75% to 74.5%
Moss et al. 2008 - -9.50% 64.5%
de los Rios-P. et al. -5.35% 12.30g -0.03% 73.97%
 Sometimes, as things get worse, they get much worse
Luo et al. 2014. Effect of
inbreeding on survival, WSSV
tolerance and growth at the
postlarval stage of experimental
full-sibling inbred populations of
the Chinese shrimp,
Fenneropenaeus chinensis.
Aquaculture 420-421:32-37

Inbreeding depression in
growth ranged from -4.16% at
F= 0.25 to -4.74% at F= 0.50.
Non-linear trend. But this is 5
generations of crossing full sibs.
Inbreeding depression for overall
survival averaged -1.27% to -1.77%
per 10% increase in F, but did not
impact WSSV survival.
Loss of Genetic Variation =INBREEDING DEPRESSION???
Not Necessarily
Sunden and Davis, 1991 Texas
Over a 4-year period, no significant loss of genetic
variability 500 founders (3 origins), 200 females and males
in each generation

Cruz et al., 2004 Mexico

Across 3 generations, no significant
loss of genetic variability. 56 founders, 26
families in G1 and 47 families in G2
Freitas et al., 2007 Brazil
Reduced genetic variability in
some hatcheries probably caused by
bottleneck effects
Perez-Enriquez et al. 2009. Genetic diversity status
of White shrimp Penaeus (Litopenaeus) vannamei
broodstock in Mexico. Aquaculture 297:44-50.

A survey of 6 hatcheries in Mexico, using 6

microsatellite loci. Even after 10+ generations of
closed breeding and an estimated inbreeding
coefficient of 26.5%, the authors found genetic
diversity and heterozygosity were higher than
expected and mean allele numbers were similar to
samples taken 5 -7 years earlier.
NEVER allow maintaining genetic diversity to take
precedent over genetic improvement. We would
not have modern day cattle, poultry or any other
livestock if that philosophy had been adopted
10,000 years ago.
 Genetic
Drift
(DERIVA):
A problem even
Ne = 1000 with a pedigree
tracking program

Ne = 50
Effective Population Sizes
Ne = (4*Nm*Nf)/(Nm + Nf)

Number of Breeding
Stock
Effective
Males Females Total Population
5 5 10 10.00
5 10 15 13.33
5 25 30 16.67
5 50 55 18.18
10 20 30 26.67
10 40 50 32.00
50 100 150 133.33
67 67 134 134.00
Establishing and Maintaining Genetic Variation
1. Founder Stock must have sufficient genetic variation
(multiple sources if possible)
2. Inbreeding -vs- drift: Small numbers can result in drift,
even if a pedigree program is in place to avoid mating
closely related individual animals
3. Keep (and use) as many breeders as possible. For
shrimp, in my opinion, at least 200 females and 200
males per generation
4. A 1:1 sex ratio increases the effective population size,
but this is difficult to enforce with L. vannamei
5. Use a pedigree program when possible, but only when
the benefits outweigh the CO$T$
6. Its often simpler and less expensive to simply
supplement or replace the breeding stock with outside
lines.
Genetics of WSSV disease resistance in penaeid shrimp
Gitterly et al., 2006 Colombia
Heritabilities for WSSV resistance were extremely
low: 0.01+0.00 to 0.04+0.02
Cock et al., 2009 Colombia
Theorized WSSV resistance would emerge as a
single gene trait (not polygenic)
Campos-Montes et al., 2013 Mexico
Heritabilities for survival. At 28 days: 0.03+0.01, at
130 days: 0.04+0.01
Caballero-Zamora et al., 2015 Mexico
Survival h2 in WSSV outbreak pond: 0.06 + 0.02;
vs. 0.00 0.02 in other ponds.
Genetics of WSSV disease resistance in penaeid shrimp
Huang et al., 2011 China
High Survival families: 22.7% + 12.3% survival after WSSV
challenge, with (23.87 + 2.56) x 106 copies g -1

Susceptible families: 0.78% + 1.70% survival after WSSV

challenge, with (65.70 + 3.44) x 106 copies g-1

Cuellar-Anjel et al., 2012 PANAMA

Survival in 3 selected families was 23%, 57% and 26% vs 0% for
Kona line. Survivors had undetectable levels of WSSV 17 days after
the challenge.
Aranguren et al., 2014 Colombia
Five generations of selection for survival. Survivals of 30.0% and
39.0% in two selected lines, vs 2.5% for a High Health line.
Survivors were all negative for WSSV according to PCR, and
exhibited no inclusion bodies in their tissue.
Genetics of WSSV disease resistance in penaeid shrimp
Huang et al., 2012 China
Survival %

Generations of Selection
Genetics of disease resistance in penaeid shrimp
How to evaluate resistance?
Different statistical models are available
Linear or threshold for individual survival at 50% mortality
Time until death?
Complex distributions of yes/no individual survival by day
Huang et al., 2011 China
We need to evaluate survival, AND viral concentrations within tissues
Or other internal damage, as in EMS
Phenotypic (Observed)Variation:
Partitioning Genetic and Environmental Components

VP = Phenotypic variance
VP = VG + VE
VG = Genetic variance (VA + VD + VI)
VE = Environmental variance

VP = (VA + VD + VI) +VE

VA = Additive genetic variance
VD = Dominance genetic variance
VI = Interactions (Epistasis)
Phenotypic Variation: Genetic and Environmental Covariance
Molecular Discrimination and Markers
* Identification
* Marker Assisted Selection
Alcivar-Warren has pioneered much of the work identifying genetic markers in
penaeids. A number of genetic maps have been developed in recent years, based
on linkage.
Perez et al., 2004 Ecuador, vannamei
51 linkage groups for males, 47 for females
Zhang et al., 2007 China, vannamei
45 linkage groups for males, 45 for females
Du et al., 2009 USA, vannamei
45 linkage groups
Wilson et al., 2002 Multi-national, P. monodon
20 linkage groups
Wang et al., 2012 China, F. chinensis
50 linkage groups for males, 44 for females

QTLs: Quantitative Trait Loci

Have influence, NOT control, over continuous traits like growth
A genetic linkage map of Pacific white shrimp (Litopenaeus vannamei): sex-linked
microsatellite markers and high recombination rates Zhang, Liusuo; Yang, Changjian; Zhang,
Yang; Li, Li; Zhang, Xiaoming; et al. Genetica131.1 (Sep 2007)

Castillo-Jurez,
Campos-Montes,
Caballero-Zamora
and Montaldo. 2015.
Genetic
improvement of
Pacific white shrimp
[Penaeus
(Litopenaeus)
vannamei]:
perspectives for
genomic selection.
Frontiers in Genetics
6:93. doi:
10.3389/fgene.2015.
00093
Universidad Autnoma
Metropolitana, Mexico D.F. y
Universidad Nacional
Autnoma de Mxico, Mexico
D.F.
6 per female
Advances in shrimp
genomics (Yu et al.,
2014; Zhang et al.,
2014) may lead to
the future
development of SNP
chips for P. vannamei,
50 per female making the
possibility of
performing GS in this
species more viable
in the near future.
100 per female
Buy a good book on genetic
improvement it doesnt have to be
mine :P
Strain effects and Genotype x Environment Interactions
are quite real in L. vannamei
HE Says: Even if its not really
broken you should be worrying (and
spending money on consultants)

I Say: If its not really broken

dont be in such a hurry to fix it.
???