wera 2008 uw
DESCRIPTION
UW WERA reportTRANSCRIPT
Overview of Shellfish Activities at
the University of Washington
Updates from Carolyn Friedman and Steven Roberts
WERA 2008
April 06, 2008
1) Population Genetics (and other)– Abalone
– Geoduck
2) Quantitative Molecular Diagnostics– OsHV - Oyster Herpes Virus
– RLP - Withering Syndrome
– QPX and related taxa
3) Physiology [Gene Discovery Efforts]– Pacific Oyster
– Abalone
– Hard Clams (& QPX) and Manila Clams
• Make changes to abalone slides indicating
that:
– Pilot scale outplant was a success
– Adult aggregation pilot
– Further outplanting work (conventional vs
habitat enriched)
– Disease/ Genetic screening
OR2 Flat OR
WA 1901
OR Flat 9
OR Flat 8
OR Flat 7
OR Flat 4
WA 1903
OR Flat 6
OR Flat 3
WA 1900
Hw a1 flat CA
OR Flat 10
WA 1916
WA 1906
WA 1924
Orf1 f lat Or
WEIRDO/ FLAT
890 White Catalina
890 White Catalina (2)
893 White Catalina
892 White Catalina
891 White Catalina
WHITE
CA 1 Pinto CA
WA 1904
AKK16 Pinto Ketchikan
Has2 assim SantaBarbara
WA 1920
Has3 assim SantaBarbara
CA 2 Pinto CA
AKK17 Pinto Ketchikan
Has1 assim SantaBarbara
WA 1898
PINTO/ THREADED
My22 Red Monterey
SMI 143 Red SanMiguelRED
UW0665 Black Carmel
UW0676 Black SNIBLACK
62132 Pink SanClemente
Mex1 Pink MexPINK
Green14 Green SanDiego
LJ7 Green LaJollaGREEN
100
100
100
69
100
70
63
68
45
55
99
62
67
57
70
73
52
33
0.01
OR Flat 2
OR Flat 1
WA 1916
CA Flat 21
WA 1918
WA 1901
CA Flat 22
CA Flat VD110
WA 1903
WA 1905
Flat/ Divergent WA
Has 4
Has 3
Has 6
WA 1904
WA 1902
Ak S 15
Has 2
Has 7
CA Pinto 1
Has 5
Pinto/ Threaded
Pink Pink lysin INTRON 2(Nathan)
62
16
10
22
11
17
70
100
99
66
0.005
Abalone
Lysin
COI
Hap C (3)
PC, PW
Hap J (1)
PA
Hap A (9)
PA, PC, PWHap E (2)
PW, PC
Hap B (8)
PW, TC
Hap I (1)
TC2
19
2
1
Hap F (1)
PC 1
Hap D (2)
PW
11
1
Hap G (1)
TC1
1
Hap H (1)
PA
1
Hap J (1)
DW?
5
1
Flat/Div.
Lysin Intron 2
404 bp
Haplotype Network
Pinto/Threaded Clade
Pintohttp://www.vetigastropoda.com
www.gastropods.com
www.gastropods.com
Flat
Biological significance of
observed
differences—fertilization studies
beginning
Abalone
All divergent pintos
from ONE location
Other abalone updates
• Pilot outplant successful! (~20% survival
after 1yr, > survival in larger size class)
• Initiated adult aggregation study
• Just funded to begin a larger scale
outplant, with hatchery rearing in
conventional & habitat-enriched tanks
Geoduck: Effects of Geoduck
Aquaculture on Wild Populations?
• Are cultured geoduck
spawning?
• If yes, what is the relative
reproductive success of
cultured geoducks?
• Are cultured geoduck
genetically distinct from wild
geoducks?
• Triploidy?
Maturation and Spawning rates
in intertidal geoducks
0
10
20
30
40
50
60
70
80
90
2 year olds 5 year olds
MATURE (%)
SPAWNED (%)
1) Population Genetics– Abalone
– Geoduck
2) Quantitative Molecular Diagnostics– Oyster Herpes Virus
– RLP - Withering Syndrome
– QPX and related taxa
3) Physiology [Gene Discovery Efforts]– Oyster
– Abalone
– Hard Clams (& QPX) and Manilla Clams
OsHV QPCR Comparison
• QPCR tests developed independently– A (UW)
– B, C, GP, and DNA polymerase gene (IFREMER)
• Goal: compare sensitivity of QPCR tests– same standard curve (purified OsHV genome)
– samples from USA, China, Japan and France
– samples with limited OsHV DNA
– similar recipes (SYBR) on same QPCR platform (StratageneMX 3000)
To learn more come to this groovy talk by: Colleen Burge….here at NSA
C CA BGPTRL IRL
UL
X
IRSUS
TRS
Oyster Herpes Virus (OsHV): Breeding
for Resistance
• Losses of seed oysters associated with OsHV in 13
of the past 15 yr in Tomales Bay, CA
• Creating potentially OsHV resistant oyster families for
outplant in Tomales Bay in 2008
• Survivors of 2003 mortality event, including high and
low performing stocks
• Families will be monitored for OsHV (QPCR) and
other diseases (histology), survival, and growth
monthly as compared to a typical hatchery stock
Pacific oyster development and survival
Goal: Develop and implement tools to study factors
involved in Pacific oyster development and survival
– Immune response (including anti-viral genes), growth, and
environmental stress (both natural and anthropogenic)
– A bioinformatic approach has been used to chose genes
and/or Expressed Sequence Tags (ESTs)
– Gene specific primers designed for Reverse Transcriptase
(RT) QPCR to examine differences in gene expression
– Compare pacific oyster tissues collected from Tomales Bay
in a year without disease pressure (2007) to Pacific oyster
larvae and seed challenged with OsHV
Funded by: National Sea Grant Oyster Disease Research Program
Work by: Colleen Burge, Carolyn Friedman, and Steven Roberts
Withering Syndrome (WS) QPCR-
Diagnostic Validation and Analytical Sensitivity
Goal: Compare QPCR
quantification to traditional
histological indices
Preliminary Results:
1) Bacterial loads in QPCR
are representative of semi-
quantitative histological
indices
2) QPCR can detect as few
as three copies of target
DNA
Flu
ore
scence
Cycle Number
5 10 15 20 25 30 35 40
30 M 3M300K
30K3K 300 30 3
Intial Copy #’s
WS: Black Abalone Disease Resistance
• Goal: Assess whetherprogeny of WS survivors aremore resistant than arenaïve or non-selectedpopulations
• Method: Challenge two groupsof black abalone to WS-RLO:
Group 1: progeny of WSsurvivors (San Nicolas Island)
Group 2: recently exposed toWS-RLO but not activedisease (ie no WS) (Carmel)
Termination Sampling Histology
0
0.5
1
1.5
2
2.5
3
META PE TOTAL
BACT
DG TOTAL
BACT
Category
Re
lati
ve
Re
sp
on
se
Carmel
San Nicolas Island
p<0.05 p<0.05p>0.05
WS Resistance Trial
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
10/4
/2006
12/4
/2006
2/4
/2007
4/4
/2007
6/4
/2007
8/4
/2007
10/4
/2007
12/4
/2007
Time (Date)
Me
an
Mo
rta
lity
(N
o.)
San Nicolas Island
Carmel
p<0.01
Black Ab RLP loads via QPCR
SNI early
Carmel late
SNI late
Carmel early
0
5000
10000
15000
20000
25000
30000
DN
A c
op
ies
/ng
Ge
no
mic
DN
A
RLP loads of Black ab Morts via QPCR
Carmel
SNI
0
5000
10000
15000
20000
25000
30000
35000
DN
A c
op
ies
/ng
Ge
no
mic
DN
A
WS: Two Novel bacteria infecting
abalone
Stippled RLONew RLO WS RLO
Analyses of novel bacteria infecting abalone
• Antimicrobial Injection study: Differential susceptibility of theWS-RLO and two new bacteria to three antimicrobial drugs
• BLAST analysis of sequences from cloned amplicon producedusing universal eubacterial primers and DNA from heavilyinfected tissue (more new bacteria than WS-RLOs in the tissue)
– WS-RLO identified in 5/96 clones
– No other known rickettsia-like bacteria amplified
– Further examination of amplified bacteria underway
S
S
R
Stippled
RLO
SRDifloxacin
RRClarithromycin
SRChloramphenicol
New
BacteriumWS RLOAnti-
microbial
New RLO cross reacts with WS-
RLO ISH probes, while Stippled
RLO does not cross react
QPX & more
Drew Hall
Cornell University
Maille Lyons and Chris Dungan
Wed 11:15
• Population Genetics– Abalone
– Geoduck
• Quantitative Molecular Diagnostics– QPCR for Shellfish Larvae
– Oyster Herpes Virus
– RLP - Withering Syndrome
– QPX and related taxa
• Physiology [Gene Discovery Efforts]– Oyster
– Abalone
– Hard Clams (& QPX) and Manilla Clams
Oyster Immune
Mon 1:45
Market size
High Mobility Group Protein
Markers ?
Summary - Field Trial
• Offspring of survivors of heavy diseasepressure are more tolerant to disease
• Mechanisms involved in host responses to P.marinus include proteases and apoptosis
• Careful selection of broodstock (potentiallyaided by markers) can benefit aquaculture andconservation efforts
Abalone, clamsand clams
Mon 9:30
Wed 11:15