The Stock Improvement objective this year continued
to evolve the breeding program to improve the efficiency
of selective breeding and performance evaluation efforts,
include new approaches to genetic line development
to address emerging industry issues, and research the
genetic basis of disease (TSV) resistance.
Pedigree
tracking and analysis of genetic diversity supported
this work, while genomics research emphasized continued
development of the linkage map for L. vannamei.
Quality control with inbreeding
analysis was delayed due
to
a water main break at Tufts University which destroyed
the majority of the genomics lab and equipment.
Fortunately,
samples were not affected
and the work
will continue
once the lab and equipment have been restored.
Work on cryopreservation
of male gametes also continued
this year as well as upgrades to the Nucleus
Breeding Center at the Oceanic
Institute to expand the capabilities
of the program.
The selective breeding
program reached an
important milestone
during FY05 in the
development of the
computerized pedigree database (Task 1.5.1).
Sufficient information
has now been incorporated that for the first
time, the database was used to draw specific
inferences
about estimated breeding value (EBV) of Consortium
lines
to a specific trait of interest using the BLUP/REML
system.
Survival
data from TSV-resistant line
shrimp subjected to multiple TSV isolates
(Task 1.2.4)
generated EBVs for TSV-resistance from Oaxaca
(0.14) and Chiapas
(0.16) that were higher than those of other
founder stocks (rng.
0.04 – 0.08). This indicates the
Oaxaca and Chiapas founders contribute the better
portion of TSV-resistance to USMSFP stocks. The information
confirmed the steady progress of the selective breeding
efforts of the Consortium for TSV-resistance since
EBVs have also increased with time.
The
computerized pedigree also
identified the effects of
inbreeding
on survival are more harsh, the harsher
the environment. For each
10% increase in inbreeding
coefficient (F),
there was found only a 3.2% reduction in
survival in
growout, while survival decreases 18 and
36 percent, respectively,
in animals exposed to TSV-A
and the
more virulent, TSV-C. The database is nearing
completion and currently
has 130,000 individual records
from
Batches
8 through 22 incorporated.
The breeding program also
continued to develop
and evaluate its TSV-Resistant (Task 1.2.2.1),
Growth (Task
1.2.2.2), Inbred (Task 1.2.2.3), and Sentinel
(Task 2.2.6) lines. Evidence is building
that selection
for TSV resistance does not adversely affect
growth, as
was previously thought. Very little difference
was found between growth of shrimp from
Batch 19 (TSV-resistant
line) and Batch 20 (Growth line) shrimp
during
FY05. This may indicate that lines can
be bred for both TSV
resistance and growth.
Plans
for development of eight
hybrid families using four
different
TSV-resistant
families and two different growth line
families
are
moving forward (Task 1.2.2.5). This initial
screening experiment should provide evidence
of the value
for hybridization of the two traits.
This possibility will
be further evaluated using the computerized
database. Plans also moved forward on
utilization of USMSFP
microsatellite markers to conduct walk-back
selection of Consortium
lines to improve selection intensity
(Task 1.2.2.1).
Blind
samples of 50 shrimp of
known pedigree
were sent to Tufts for testing and
identification of
parentage.
If identification is 100% accurate,
the program will begin
transition from
the use of physical
tags to genetic
tags for family identification in its
selection efforts. Although
pedigree tracking and
allele association
analyses
were slowed due to damage of Tufts
facilities, work was completed
for the 50 families
of Batch 19 shrimp
received in July 2004 and challenged
with the three TSV serotypes
(Task 1.2.2.1).
Research on the genetic
basis of TSV resistance
also reached significant
milestones
during
FY05. The program
established a bioinformatics database
and search capability to identify genes
associated
with
traits of economic
importance (Task 1.2.3.1). Approximately
5,000 ESTs were entered into the Tufts’ database (ShrimpESTbase).
A
first-generation, microsatellite-based,
low-density linkage map (ShrimpMap)
was also produced that can
be used for mapping QTLs of economic
importance,
and construction on a second-generation
map based on ESTs
and SNPs was begun (Task 1.2.3.2).
DNA from the shrimp mapping
families is available to
any researcher interested
in contributing markers to the development
of a high
density linkage map.
A
total 100 microsatellite
primer sets
are being tested for polymorphism
with the mapping
panels. Primers that did not show
polymorphism with the USMSFP
linkage mapping family
may be applicable
to other domestic breeding programs
with different gene pools,
and are currently available
for industry
use.
A genome-wide scanning
was also initiated
to identify candidate
QTLs associated with
resistance to TSV with
14 markers geneotyped thus far. The program is
currently
at a critical crossroad in linkage mapping development
to determine if additional families are needed
to supplement the existing
LMRF and if RMFs for
QTL analysis are
appropriate candidates. Addition of new families
with higher levels
of polymorphism may
help speed up linkage
map development.
Work on cyropreservation
of gametes re-initiated
last year continued
during FY05 by profiling
sperm morphology
and egg water-sperm reaction studies by stabilizing
testing conditions (Task 1.1.2). Apparent differences
were found in morphology of chromation fibers.
Research is currently
ongoing to determine
if those changes
were artifacts of the cyropreservation process.
Changes in the thelycum
after mating are also
being documented,
which may prove important in obtaining high fertilization
after cyropreservation.
Disease Control
Disease research continued to take a proactive
approach to protect the industry by improving the
precision
of diagnostic methods and tools, and gaining insight
into the mechanisms and transmission of disease.
Particular
attention this year was again focused on TSV and
NHP due to continued industry
concern, and infectious myonecrosis
virus (IMNV), an emerging and significant disease
of
L. vannamei that has not been identified in the
US, but has been added to
the USMSFP working list of
specific
(C-1,2) pathogens. The USMSFP has recommended IMNV
be included on the OIE list of notifiable pathogens.
For the past four years, the USMSFP has continued
to develop its TSV- resistant line of animals for
the
domestic broodstock industry and evaluated the
effectiveness of technology transfer.
During
FY05, TSV challenge studies
of commercially available
families (Task 1.2.4.1)
indicated USMSFP stocks have had a significant
impact on development of
US broodstock companies,
which continue
to rely on Consortium stocks for their base. It
is now common to find commercial
companies producing families
that exhibit up to 100% resistance to TSV (A serotype).
US broodstock companies have made tremendous inroads
in selling product both in the US and overseas.
It
can be estimated that L. vannmaei now accounts
for 40% of all shrimp produced
in Thailand, Vietnam, Indonesia,
and India, up from less than 10% in 2002.
While work continues on
TSV line development
for industry, research
has evolved to utilize
Consortium lines as
a tool to understand the genetic and physiological
basis of disease resistance in shrimp. Results
on the
mechanisms of TSV resistance (Task 2.5.1.4) confirmed
suppositions made last year that TSV pathology
and resistance is related
to viral loads. TSV-resistant
animals apparently do not prevent a build-up of
levels
of the virus in their bodies; rather they build
up levels much more
slowly than non-resistant
animals.
The
same level of virus was found
to kill both resistant and
non-resistant
animals, and all
animals have some
viral load when exposed. Work last year allowed
the Consortium to
transition to TSV load instead
of survival
as the key metric for evaluating response and
examining
the genetic basis of disease resistance. Other
research indicated
an additive effect
of the pesticide,
endosulfan, on mortality
of TSV-infected shrimp (Task
2.5.1.5). Mortality
increased in both TSV-infected
and non-infected shrimp,
but was independent of molting
stage.
Work also continued this
year on the multi-institutional
collaborative effort of the USMSFP in Texas
to discern the causes
and origins of a TSV
outbreak
that occurred
in early June 2004. The viral strain (04 USTX)
is closely linked phylogenetically to the Southeast
Asian group
of TSV isolates, but there have previously
been no connections
to its origins.
A
particularly significant
finding this year was identification
of a linkage
between
the Asian group with isolate 98-HO (Task
2.3.1.1.1).
This indicated that TSV was brought to Asia
from
Honduras. As of this writing, samples taken
from south Texas
during 2005 did not contain TSV (Task 2.2.4),
indicating no genetic persistence of the
disease. Sampling
will continue on specific sites, with additional
funding
secured through APHIS.
Significant progress was
also made on the epidemiology
and transfer mechanisms of NHP. Research confirmed
that multiple strains among geographic isolates
of NHP exist based
on sequences from the
gryB region of
the genome (Task 2.3.1.2.1), and that NHP can be
transferred through
culture water using
the USMSFP chronic
infection
assay method (Task 2.4.3.2.2). There appears a
flagellated form of
the bacterium, indicating
a mode of transmission
independent of a specific host or vector. However,
it was also found that the infection is not transferred
by co-habitation with infected live shrimp (Task
2.4.3.2.3).
Cannibalism of dead shrimp caused infection and
transmission was lowest
at the lowest (10 ppt)
and highest (40 ppt)
salinities tested. Survival was independent of
salinity.
The USMSFP continued efforts
on two diseases that
are not present in the US, but which have potential
to
spread rapidly if diagnostic and prevention methods
are not in place. Rapid progress has been made
on IMNV, which was
first characterized
last year by
the USMSFP.
Additional detection methods for IMNV were developed
during FY05 (Task 2.3.1.1.6), one of which — a
highly specific nested PCR — was transferred
to IntelliGene Corp. for commercial application.
Challenge
studies against IMNV were
also initiated with 42 families
from Sygen International (Task 1.2.4.1). Survival
of these families ranged from 20 to 100 percent,
indicating
the potential for development of stocks resistant
to this rapidly emerging and devastating disease.
Challenge
models developed last year for sprioplama penaeii
also were used to determine that L. vannamei were
much more
susceptible than L. stylirostris to this bacterium
(Task 2.4.3.1).
The need to remain vigilant
has also been highlighted
by the number of new diseases and new strains of
existing ones that
appear to be emerging.
During FY05, outbreaks
of WSSV were identified in Guam, Brazil, Mexico,
and
Saudi Arabia. Some TSV variants arose in Venezuela
and Indonesia, and TSV-C was detected in Nicaragua
(Task 2.2.2). NHP was also identified in Jamaica.
New
diseases found included haplosporidian and intranuclear
mirosporidian in L. vannamei in Central America,
a new enteric parvovirus
in Fennenopenaeus indicus
and
Penaeus monodon in East Africa, and a new HPV strain
in P. monodon from Southeast Asia and East Africa.
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