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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