Enhancing Stress Resistance of C lt d Cl Th h T i l id Cultured Clams Through Triploidy:

Final Report on Field Trialsand Laboratory Challenges

John Scarpa Harbor Branch Oceanographic Institute @FAUJohn Scarpa, Harbor Branch Oceanographic Institute @FAUShirley Baker, UF, SFRC, Fisheries and Aquatic Sciences

Leslie Sturmer, UF Cooperative Extension Serviceh k d F D f F d d EChuck Adams, UF Department of Food and Resource Economics

Hypothesisyp

• Mortalities from f msummer stressors can be reduced by creating sterile clams through sterile clams through triploidy

•

Triploidy = 3 sets of chromosomes

Why triploidy?Why triploidy?

Triploids divert energy • Triploids divert energy from reproduction to storage and growthstorage and growth

• Used in PNW oyster aquacultureaquaculture

• Need for hardier clam strain in SE

Specific objectivesSpecific objectives

• Create replicate diploid/triploid families• Compare growth & survival during grow-outCompare growth & survival during grow out• Compare physiological responses to stress• Examine economicsExamine economics

Meiotic and Mitotic Stages in Eggs of Mercenaria mercenariaEggs of Mercenaria mercenaria

Sperm

Metaphase I Early anaphase I PB 1Late anaphase I

PB 2 Pronuclei Syngamy 1st Cleavage(2-Cell stage)

Mean Time to Formation of First Polar Body (PB 1), Second Polar Body (PB 2), and First Cleavage in Hard Clam EggsPolar Body (PB 2), and First Cleavage in Hard Clam Eggs

60

e

40

50

each

sta

ge

30

40

fcel

ls a

t e PB 1PB 21st cleavage

10

20

enta

ge o

f 1st cleavage

0

10

0 3 9 5 31 40 60 80

Perc

e

0 13 19 25 31 40 60 80

Time (min) post-insemination

Flow CytometryF ow ytom try

Polar Body 2 releasey

Polar bodies Flow Cytometry outputbodies

Male genomeMale genome

Cytological and Flow-Cytometric Data from Triploid Induction Experiments

Pre-PBII

Post-PBII

Pre-fertilized Pre-PBI

Trial #, treatment

Cytological Data (% of fertilized eggs)Triploid

(%)

PBI 50 7 43 39PBII 3 30 67 0

PBI 83 2 3 0PBII 23 77 0 0

55

5

1

2 PBII 23 77 0 0

PBI 100 0 0 0PBII 0 100 0 0

PBI 90 10 0 93

13

PBI 90 10 0 93PBII 0 44 56 33

PBI 85 0 0 33PBII 28 72 0 83

5

5

4

0

PBI 55 0 21 77PBII 56 39 5 86

PBI 41 0 1 48

66

PBI 41 0 1 48PBII 66 26 8 57

PBI 69 2 8 26PBII 36 48 13 69

5

48

7

Juvenile Growth Studies

Growth (%) in shell length (A) and live weight g g(B) of juvenile triploid Mercenaria mercenaria

80

90

th)

DiploidPB 1 triploid

a b bA400

450

ght)

DiploidPB 1 triploid

a b cB

40

50

60

70

ease

in S

hell

Leng

t pPB 2 triploid

a b b200

250

300

350

400

ease

in li

ve w

eig PB 1 triploid

PB 2 triploid

10

20

30

40

GR

OW

TH (%

incr

e

a b b

a a a

0

50

100

150

200

RO

WTH

(% in

cre

a b c a b b a a b

01 2 3 Total

TIME (weeks)

01 2 3 Total

TIME (weeks)

GR

Growth (% live wt) of juvenile triploid M i i i i t t900 Mercenaria mercenaria in experiment two

700

800

900w

eigh

t)

500

600

700

ase

in li

ve w

300

400

500

H (%

incr

ea DiploidTriploid

200

300

GR

OW

TH

0

100

K M O AllK M O All

FAMILY

Survival under stress• 45 mm SL

Temperature: 90°C• Temperature: 90°C• Salinity: 10, 25, 40 ppt• Oxygen: Normoxia or hypoxia• Oxygen: Normoxia or hypoxia

M lMortality

Survival – 10 and 40 pptSurvival 10 and 40 ppt100 100

10 ppt 40 ppt

60

80

tive

Surv

ival

Diploid, 10ppt, Normoxia

Triploid, 10ppt, Normoxia

Diploid, 10ppt, Hypoxia

Triploid, 10ppt, Hypoxia60

80

tive

Surv

ival

Diploid 40ppt Normoxia

0

20

40

% C

umul

at

0

20

40

% C

umul

at Diploid, 40ppt, Normoxia

Triploid, 40ppt, Normoxia

Diploid, 40ppt, Hypoxia

Triploid, 40ppt, Hypoxia

01 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Time (Days)

01 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Time (Days)

At li it t t i l id l h • At salinity extremes, triploid clams have no advantage over diploid clams

Survival – 25 ppt

80

100l

60

80

ve S

urvi

va

40

Cum

ulat

iv

Diploid, 25ppt, Normoxia

Triploid, 25ppt, Normoxia

Di l id 25 t H i

0

20% C Diploid, 25ppt, Hypoxia

Triploid, 25ppt, Hypoxia

01 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Time (Days)

Triploidy may increase survival in hypoxia

Physiological response to stressy g p

• Oxygen uptake ratesyg p• 50 mm SL• 25 ppt or 15pptpp pp• 68, 77, 80, 86, and 90°F

Oxygen uptake rate –temperaturetemperature

) Diploid

n (u

g/g/

h)

• Metabolic rate increases with

Triploid

nsum

ptio

n increases with temperature, but not above 80°F

oxyg

en c

on

• Triploid clams h d

Log

o have no advantage over diploids

68 77 80 86 90Temperature (°F)

Oxygen uptake rate – salinityOxygen uptake rate salinity

• Triploid clams have /h)

• Triploid clams have lower metabolic rate at 15 ppt than at 25 ti

on (u

g/g/

ppppt (80°F)

l d l

cons

umpt

• Triploid clams may use less energy during stressg

oxyg

en

25 ppt 15 ppt stressLog pp pp

Comparison of Survival and Hsp70 Synthesis in Triploid and Diploid hard clams under Normal and Heat Shock Conditions.

(Sampling times: 0T (day 10), 4 hr, d1, d5 or d7) End of

experiment5-7 days0 TimeDiploids

10 daysStart of

Heat shock

25°0C 25°CAcclimation

25°CControl

35°CAcclimation

25°CHeat Shock

35°C

Acclimation Control

Triploids

25°C 25°CAcclimation

25°CControl

35°CAcclimation

25°CHeat Shock

35°C

Experimental System(1) Acclimation Unit : Sump connected to a chiller @ 25±1o C.( ) cc at o U t Su p co ected to a c e @ 5 C(2) Cold Treatment Unit: Sump connected to a chiller @ 25±1o C.(3) Hot Treatment Unit: Sump with titanium Heater @ 35±1o C.

(1)

(2)

(3)

Hsp70 and Survival (± SD) in Triploid and Diploid Hard Clams (SL=31.6 ± 6.6) Following Severe Heat Shock (25-35oC).200

100

150

200

µg T

SB-1

)

3N

0

50

100

sp70

(RU

µ

2N

0T0 4h D1 D7H

s

Time after heat shock

6080

100

val (

%) 3N-M

2N-N

0204060

Surv

iv

00 1 2 3 4 5 6 7

Time (days) after heat shock

Survival (± SD) of hard clams (SL= 44.0± 3.3) exposed to severe heat shock (25-35oC) was influenced by genetic background represented by higher

initial Hsp70 concentration (P= 0 042) than by ploidy (P= 0 184)initial Hsp70 concentration (P= 0.042), than by ploidy (P= 0.184).

90100

)

3N-K 2N-L

100

)

3N-M 2N-N

405060708090

Surv

ival

(%)

405060708090

Surv

ival

(%)

3040

T 0 Day 1 Day 2

Time following heat shock

3040

T 0 Day 1 Day 2

Time following heat shock

90100

3N-O 2N-P

200

250

SP-1

)3NA

B

Aa a

4050607080

Surv

ival

(%)

50

100

150

Hsp

70 (

RU

µg

TS 3N

2N

A

a a a a

3040

T 0 Day 1 Day 2Time following heat shock

0K & L M & N O & P

H

Family

GROW-OUTGROW OUT

Leslie SturmerLeslie Sturmer

Clam samples

Hurricane Charley

Mean values of different parameters measured for PB2 triploid clams cultured in Cedar Keyfor PB2 triploid clams cultured in Cedar Key

Diploids Triploids T-testN Mean SD N Mean SD Signif.g

Shell Length 32 23.4 ± 3.5 13 19.7 ± 3.2 0.002Shell Width 32 11.3 ± 1.7 13 9.3 ± 1.5 0.000Live Weight 32 3 33 ± 1 32 13 1 97 ± 0 85 0 001Live Weight 32 3.33 ± 1.32 13 1.97 ± 0.85 0.001

Dry Meat Weight 18 0.104 ± 0.039 7 0.068 ± 0.026 0.034Condition Index 18 5 6 ± 0 5 7 6 6 ± 0 5 0 000Condition Index 18 5.6 ± 0.5 7 6.6 ± 0.5 0.000

Four hurricanes hit Florida in 2004 and destroyed 80% of all clams planted for the study. Data presented is from only one group of clams cultured in Cedar Key and sampled in December 2004. Triploid clams were

ti t d t 42 70% b f th h i b t l 29% ft T i l id l i ifi tl ll festimated at 42-70% before the hurricanes, but only 29% after. Triploid clams were significantly smaller for all parameters measured except condition index. Histological analysis indicated 50% of diploid clams had spawned, whereas 100% of triploids had no mature gonad.

Grow Out 2 (PBII triploids)Grow Out 2 (PBII triploids)

Ploidy Length Weight Cond. Survival Cedar Key

(mm) (g) Index (%)

2N 45.9 30.6 4.77 80.13N 45.9 29.0 5.30 69.4

Ploidy Length( )

Weight (g)

Cond. Index

Survival (%)

Charlotte Harbor

(mm) (g) Index (%)

2N 50.8 43.8 4.65 48.6

3N 48.2 33.9 4.65 43.1

Economics (Cost Categories)• Broodstock Conditioning: 2x # of clams

(capital investment, not calculated)Spawnin : increase area? cost?• Spawning: increase area?, cost?

• Chemical Treatment: CB/DMSO ~$20.45/5M eggs (= $0.02-0.04/1K 1mm seed)gg ( $ )

• Chemical Waste Disposal: ~$71.20/5M eggs(= $0.014-0.028/1K 1mm seed)Tripl id V rific ti n: $100/5M s • Triploidy Verification: $100/5M eggs (= $0.10-0.20/1K 1mm seed)

• Larvae Culture: no sign. expense anticipatedg p p• Setting: no sign. expense anticipated• TOTAL = $0.14-0.27/1K 1mm seed or ~5-10%

inincr.

SUMMARYSUMMARY• Produced triploid clams • Growth of triploids in lab was lower• Growth of triploids in lab was lower• Growth of triploids in field was

similar/lowersimilar/lower• Survival/stress resistance of triploids in lab

exhibited mixed results (Hsp70 selection)• Survival of triploids in field was lower• CI of triploids in field was similar/greater • Cost of producing triploids is minimal• No apparent advantage of triploid clams for

Fl id lt i tFlorida culturists

THANKSTHANKS totoFlorida Sea Grant (R/LRFlorida Sea Grant (R/LR--AA--39) and USDA support 39) and USDA support Florida Sea Grant (R/LRFlorida Sea Grant (R/LR AA 39) and USDA support 39) and USDA support

Many, many people who assisted in collecting, Many, many people who assisted in collecting, caring, experimenting, and analyzing clams:caring, experimenting, and analyzing clams:caring, experimenting, and analyzing clamscaring, experimenting, and analyzing clamsTony Heeb (Cutthroat Clams)Tony Heeb (Cutthroat Clams)Roy Kibbe (Kibbe Clams)Roy Kibbe (Kibbe Clams)Dan Leonard (Bull Bay Clams)Dan Leonard (Bull Bay Clams)Dan Leonard (Bull Bay Clams)Dan Leonard (Bull Bay Clams)Chris Taiani (Cedar Key)Chris Taiani (Cedar Key)Terry Lange (Ft. Pierce)Terry Lange (Ft. Pierce)Eric Cassiano, Elise Hoover, Kerry Weber (UF)Eric Cassiano, Elise Hoover, Kerry Weber (UF)Kyrstal Baird, Fred Prahl, Chris Withstanley Kyrstal Baird, Fred Prahl, Chris Withstanley (HBOI)(HBOI)(HBOI)(HBOI)Eman ElEman El--Wazzan (FIT)Wazzan (FIT)and those we have missedand those we have missed

Questions?Questions?