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