Tolerance of different hard clam stocks to

various isolates of Quahog Parasite Unknown (QPX)

Soren Dahl, Mickael Perrigault, and Bassem Allam

Marine Sciences Research CenterStony Brook University

New York

Protistan parasite of Mercenaria mercenaria

• Important fisheries, aquaculture; U.S. East– 1st Industry wide disease concern, near market size clams

• Mortality events; Cultured & Wild clams– 1960’s New Brunswick Canada– Discovered in Virginia, 1997

• Thraustochytrid: common osmoheterotrophs

• Heavy mortalities (up to 100%) but also present at extremely low prevalence (>0.1%) in apparently healthy clam populations

• Opportunistic parasite– Disease/mortality disadvantaged groups

Possible sources of variabilityIn the host• Susceptibility varies due to

geographic origin of broodstock (VIMS, HSRL)

In the parasite• Pathogen range; unknown variation

• Diverse mortality events– Diagnostic presentations; lesion sites, disease severity

• Morphological or physiological differences among QPX isolated from different locations (Buggé & Allam, in prep.)

• Potential differences in pathogenicity

Hypothesis: Prevalence and severity of QPX infections in Mercenaria mercenaria varies significantly due to particular interactions between clam stocks and QPX organisms.

Objectives: 1. Compare tolerance of different cultured

hard clam stocks to QPX infection.2. Compare the pathogenicity of QPX

organisms geographically or morphologically distinct

QPX Transmission Approach: Laboratory

Investigate susceptibility of different clam strains to

different QPX isolates • Used a recently-developed experimental transmission

method by injecting QPX into clam’s pericardial cavity (NSA 05; Dahl and Allam, submitted)

• Three QPX Cultures: Isolated from infected clams collected in:

– Massachusetts (MA-1), New York (NY8BC7 and NY20AC5)• Minimal Essential Media: control• 4 strains of naïve seed (~1yr):

– Massachusetts– Virginia – New York– Florida

• Histology sampling; 15 & 27 weeks– Prevalence: # of positive individuals

• Mortality monitoring• Moribund samples

MA-1, NY-1, NY-2

0

10

20

30

40

50

60

70

80

MA NY VA FL

MEM

A

B

C

Final cumulative mortality (27 weeks)

% m

orta

lity

Seed type

05

10152025303540

MA NY VA FL

% m

ort

alit

y

A

B

C

Adjusted by amount of control mortality

% m

orta

lity

Seed type

00 0 2.5

7.5

2.5

2.1

16.7 15.032.2

5.6

20.9

0

20

40

60

80

MA NY VA FL

15 weeks

27 weeks

Moribund

4.1 0.0

22.5 25.0

45.0

20.8

17.9

39.3

45.0

54.851.8

51.8

0

20

40

60

80

MA NY VA FL

Isolate: NY-1

0.0

12.5 15.1

37.5

11.915.4

24.4

8.3

26.435.4

25.0

25.0

0

20

40

60

80

MA NY VA FL

Isolate: NY-2

Isolate: MA-1 Percent prevalence of infection by isolate.

Low

High

Intermediate

Sign. @ p<0.05 , William’s correction

QPX prevalence can be correlated to the type of clam inoculated, stronger significance is attributed to the isolate

of QPX that was injected into the clam

Statistical Analysis: Test independence of prevalence data, Contingency tables (frequency independent of variables)Log-linear analysis, 3 way tables: Seed Type, Isolate, Infection

*p<10-5Simultaneous interaction of all three variables: NS

Two way interactions: S SSeed Type: S*Isolate:

G-test of independence for 2 way tablesIndependence of Seed type NSMA-1: FL ↑SNY-1: VA ↑SNY-2:

SIndependence of Isolate S**FL:MA: SNY: SVA:

**High NY-1; p<10-4Lack of prevalence in MA-1 treatment

Multiple Isolate injection trial summary

• MA-1: least pathogenic – Low mortalities, prevalence

lacking

• NY-1: most pathogenic– Consistently high prevalence,

severe mortalities

• NY-2: more pathogenic than MA-1 but not quite as virulent as NY-1

– Intermediate prevalence, mortality often rivaled NY-1

•MA: most tolerant – Low mortality and prevalence

•NY: variable– Moribund prevalence; highest

(NY-1), lowest (MA-1) – Not as susceptible as FL or VA,

not as tolerant as MA

•VA: infection from all 3– Most often prevalent, typically

high

•FL: consistent range– MA-1: low, NY-1: high,

NY-2: middle– more susceptible than MA or NY

QPX Isolate Hard clam type

QPX Transmission: Field approach

Aim: Infective pressure and dynamics in environmentCompare the susceptibility of different clam

strainsInvestigate early stages of the disease

Raritan BayFlanders BaySouthold BayNorthwest Hbr

Method: Deploy naïve seed clams in cages placed on bottom;3 strains of clams: New York White, New York notata,Florida

4 sites; 3 to 5 replicates/site500 seed/bag

•QPX prevalence and clam mortality

Gill

MantleVisceral mass

Foot

Siphon

QPX presentonly in mantle

QPX present in

~85% pallial organs

N=60

N=120N=48

Peconic estuary field deployments Similar sampling schedule: Diagnosis of all 3 sites are...

Good News!

Relieve fears of epizootic•Historic Transplant sites•Previous detection at very low levels•Wild populations

Assay validated in Raritan Bay

Negative

Conclusions

• QPX tolerance of hard clams can be correlated to the geographic origin of the broodstock. A gradient of resiliency can be described as a latitudinal trend; higher tolerance in clams from the north.

• Intensity and severity of infection can be heavily dependent on the virulence of a particular QPX strain

• Pallial organs represent a portal of entry for the parasite and disease can develop within weeks

Conclusions

• Genetic or Poor Acclimation?– Lab controlled stable environment; 20°C – Transport stress; injected

Physiological differences based on stock, influencing resistance

Equally Disadvantaged

• Population selection by parasite– QPX not in the south; less tolerant of high summer

temperatures than clam (in vitro results)

• Restoration applications need caution in stock selection– Previous QPX activity– Identification of resistant stocks– Local/Regional selection processes

Not all Quahogs are created equal

Not all QPX are created equal• Potential threat of disease is determined

by virulent capabilities of the QPX strain– Local activity: naïve seed assay, isolate strain

Acknowledgements

• New York Sea Grant• New York State D.E.C., Marine Resources• Cornell Cooperative, Southold, NY

(S.P.A.T.)• Hatcheries, VIMS• Marine Animal Disease Lab:

– Tech, Follow students; ‘QPX-Clan’

• Faculty and Staff: MSRC, SBU

Questions?

Thanks for Listening!

See you at the…

Temperature (ºC)

Per

cent

gro

wth

(%

)

0

20

40

60

80

100

0 10 20 30 40

MA

NY 1

NY 2

Factors affecting QPX growthIn vitro growth of QPX cultures: optimal around 20-23° C (different for different isolates), in agreement with QPX

prevalence in the field

0%

20%

40%

60%

80%

100%

NY Moribund

MA-1

N=1

NY8BC7

N=27

NY20AC5

N=6

0%

20%

40%

60%

80%

100%

MA Moribund Heavy

Moderate

Light

Rare

MA-1

N=5

NY8BC7

N=4

NY20AC5

N=8

0%

20%

40%

60%

80%

100%

VA Moribund

MA-1

N=10

NY8BC7

N=13

NY20AC5

N=9

0%

20%

40%

60%

80%

100%

FL Moribund

MA-1

N=3

NY8BC7

N=25

NY20AC5

N=10

Log-linear analysis for three way tables: Testing independence of Variables: Seed Type (4 classes); Inoculate (3 classes); Infection Status (2 classes). Probability of data, for a sample, resulting from model with a term removed.

The degree of association between any pair of variables would depend upon the different levels of a third. For each class of Infection Status, Inoculate and Seed Type are associated.

Within a particular inoculatetreatment the clam type andinfection status are associated. Given a class of seed type there is a consistent association of inoculate treatment and infection status.

More substantial P values represent a greater source of influence on the results.

QPX prevalence can be correlated to the type of clam inoculated, but is even moreattributed to the isolate of QPX that was injected into the clam

    P values

Interaction term: T-1 T-2 Moribund

Three way   0.11 0.23 0.33

SeedType X Inoculate 0.45 2.52 10-9 6.04 10-5

SeedType X InfectionStatus 0.02 0.01 0.51

Inoculate X InfectionStatus 2.28 10-8 0.02 4.25 10-6

Factor term:  

SeedType   0.13 4.13 10-11 0.0001

Inoculate 1.75 10^-

6 6.54 10-11 1.66 10-10

InfectionStatus 5.60 10-9 0.0006 1.12 10-5

Complete Independence 5.20 10-7 3.71 10-12 9.75 10-10

     

  T-1 T-2 Moribund  

Inoculate  P Major 2nd P Major 2nd P Major 2nd

MA-1 NS low %’s   NS low %’s   NS = 6-32%  

NY8BC7 NS =21-45%   0.0344 FL, >   NS = 45-55%  

NY20AC5 0.0188 VA, >   NS low %’s   NS = 25-35%  

   

T-1 T-2 Moribund  

 Seed type P Major 2nd P Major 2nd P Major 2nd

MA 0.0213 A, < B, > NS <   NS = 21-45%  

NY 0.0038 A, 0   NS <   0.0002 A, < B, >

VA 0.0043 A, < C, > NS = 17-24%   NS = 32-52%  

FL 3.4 10-5 B, > A, < 0.0107 A, < B, > 9.5 10-5 B, > A, <

Inoculate treatment: A= (MA-1), B= (NY8BC7), C= (NY20AC7).

Tests of independence: Row by Column (RxC) contingency table (2 way), G-test (William’s correction) & Gabriel's simultaneous test procedure; all maximal non-significant sets of rows and columns

More P values are significant due to the isolate treatment.

Test for independence of Seed type and Infection status within Inoculate

Test for independence of Inoculate and Infection status within Seed type

Cytotoxicity of different QPX isolatesH

em

ocy

te v

iab

ility

(O

D 5

60

nm

)

0 .0 0

0 .0 2

0 .0 4

0 .0 6

0 .0 8

0 .1 0

0 .1 2

0 .1 4

0 .1 6C

ontr

ol

MA

2

20A

C2

8BC

7

Con

trol

MA

2

20A

C2

8BC

7

Con

trol

MA

2

20A

C2

8BC

7

Con

trol

MA

2

20A

C2

8BC

7

Oyster NY clam FL clam MA clam

In vitro

In vitro toxicity varies among QPX isolates

Mortality of Florida Seed

0

10

20

30

40

50

60

70

1 21 41 61 81 101 121 141 161 181day since injection

% c

umul

ativ

e m

ort

alit

y

FL;a

FL;b

FL;c

FL;e

T-1 sample at day 105A

B

Prevalence: Florida seed clams

15.1

45.0

2.515.4

2.1

39.3

26.4

51.8

15.0

0

20

40

60

80

MA-1 NY8BC7 NY20AC5Treatment

Per

cen

t P

reva

len

ce

T-1; 3.8 months

T-2; 6.8 months

Moribund

Most definitivedisplay of trends as seen in other seed type results,as well as the results of in vitro pathogenicity.

1960’s NB1989 PEI

1990’s MANY 2002

1976 NJ

1997 VA survey

2003 NY, RI

QPX disease; History andGeographic distribution

1990’s NSsurvey