Bay Scallop Genetic Resources and Applications

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Overview and Application of Bay Scallop Genomic

ResourcesSteven Roberts

Marine Biological Laboratory

Woods Hole, MA USA


Bay Scallop

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-fishery product-aquaculture product-scientific model

-muscle biology-photoreception

-essential environmental component

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Outline• Metamorphosis

– DEG– ESTs (overview) [example Iodothyronine Deiodinase]

• Muscle Growth– Candidate Gene [example Myostatin]

• Other uses for ESTs– Environmental response– Population genetics and restoration evaluations

• Available markers


MetamorphosisTrocophore -> D-hingeBetween day 9-20 larvae first become “competent” then metamorphose into adult form.

High Mortality


Why study bay scallop metamorphosis?

• Evolutionary Perspective

• Determine suitable natural habitat for successful recruitment

• Baseline information for identifying abnormalities

• Optimize aquaculture production


Approach

Comparative transcriptomic analysis of developing bay scallops prior to (D-hinge), during (pediveliger), and following metamorphosis (spat)


Differentially Expressed Genes (DEG)

Primer Pair BPrimer Pair A Primer Pair C


Differentially Expressed Genes (DEG)

“D”

hing

e

pedi

velig

er

spat

“D”

hing

e

pedi

velig

er

spat

** **

ArbitraryPrimers:

MW

ACP 22 ACP 29

“D”

hing

e

pedi

velig

er

spat

“D”

hing

e

pedi

velig

er

spat

ACP 27 ACP 28

a

bc

a= Heat shock protein 70

b= serine protease precursor

c= pheromone receptor


Expressed Sequence Tags (ESTs) agtacgtaccgatagcatgatgcatgcatgctagcagcattacgagctagcgcattcgcaagctaagtacgtaccgatagcatgatgcatgcatgctagcagcattacgagctagcgcattc

ttcgctgacacagtcgacagtcnagtacgtaccgatcgcgcgccagcatgatgcatgcatgctagcagcattacgagctagcgcattcgcaagctaagtacgtaccgatagcatgatgcatgcatgctagcagcattacgagctagcgcattc

tcgctgacacagtcgacagtcagcatgatgcatgcatgctagcagcattacgagctagcgcattcgcaagctaagtacgtaccgatagcatgatgcatgcatgctagcagcattacgagctagcgcattcgcaagctannnggttctcgttgattcgctgacacagtcgacagtcn

mRNA cDNA

Data Processing

mRNA cDNA

mRNA cDNA


ESTs

• Individual clones sequenced– D-hinge 671– Met 61– Set 932– Adductor 367– Gonad 58

• 8% sequences <200 bp• Average length - 719 bp• 970 singletons• 217 contigs


ESTs

• A. irradians – ESTs 7057 – CoreNucleotides 85

• Contigs 845• Singletons 2746• Unique Seqs 3591

C.gigas granulocyteZap Express cDNA library

- 2646 ESTs sequenced and annotated

- 343 contigs and 1319 singletons

-1662 Unique Seqs

- >40% not identified previously


Hind limbs, rapid differentiation

Fore-limb emergence to complete tail reabsorption

Acquisition of hypothalamic sensitivity to thyroid

hormones

Premetamorphosis Prometamorphosis Climax Postmetamorphosis

T3/T4

TSH

TRH

Frog Metamorphosis


Iodothyronine Deiodinase

Type 2 ID enzyme required for production of active

thyroid hormone• Tissue Distribution in Adult Bay Scallops

– Endostyle

Mucus secreting pharyngeal organ that facilitates filter feeding. Iodine concentrating ability and can synthesize thyroid hormone.


T-iso

Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

CCMP459

T-iso

T-iso

T-isoT-iso

T-iso

T-iso T-isoT-iso T-iso

CCMP459

T-iso

CCMP459

T-iso

CCMP459

T-iso

CCMP459

T-iso

CCMP459

T-iso

T-iso

Deiodinase u (ai Du)

Deiodinase v (ai Dv)


Interrelationship with External Factors

Diet: Pavlova (e.g. CCMP549) has over 3 times more (%) phenylalanine than Isochrysis

Differential uptake by algae of selenium. Selenium required for synthesis of deiodinases.


Does myostatin function in a similar manner in shellfish as it does in mammals?

= ==


Myostatin expression in fish


Scallop Myostatin

0

50

100

150

200

250

300

Mantle Gonad Heart DigestiveGland

Gill Muscle

Tissue

Hea

rt 1

Hea

rt 2

Gill

1

Gill

2

Dig

estiv

e G

land

1

Dig

estiv

e G

land

2

Gon

ad 1

Gon

ad 2

Man

tle 1

& 2

Mus

cle

1 &

2

Real-time RT-PCR – Dual labeled probes

Northern Blot

At least 6 times higher expression in adductor muscle tissue


Myo-Blast Experiment

Figure 5. PCR products from RNA from adult scallops (N=2) treated with MyoZap and not treated (contols). Differentially expressed genes are indicated with lower case letters.

Cyclin T


Myo-Blast Experiment

Control Experimental


Other uses of ESTs


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Enhancement

Broodstock

Condition

Spawn

xx


SeedWellfleet

ChathamDennis

32101


EST-SSR M26Wellfleet

ChathamDennis

40

1030

Falmouth

Chilmark

20km


Wellfleet

Dennis

32101

Chatham

g340

m26

n391

s336

Diver (aug) Diver (nov) Diver (nov) Spat Bag


Regional

N391G340

M26

N391G340

M26


Other Bay Scallop Markers

• EST-SSRs

• “Conventional” Microsats (Poster)


EST-SNPs


Acknowledgments• USDA-NRI 2003-35206-12834• Barnstable County, MA - CCCE• Linda McCauley & Christina Romano

• TECHNICAL [mbl]

• Hyun-Woo Kim & Don Mykles• MYOSTATIN [csu]

• Chris Dickson & Adam Bisonette• MYOSTATIN [undergraduate students]

• Gary Wikfors & Mark Dixon• METAMORPHOSIS [nmfs]

• Gabriele Gerlach• GENETICS [mbl]

• Steve Tettlebach & Maria Kretzman• GENETICS New York [liu]

Bay Scallop Genetic Resources and Applications

Technology

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