1. Tolerance of different hard clam stocks to various isolates of Quahog Parasite Unknown (QPX) Soren Dahl, Mickael Perrigault, and Bassem Allam Marine Sciences Research Center Stony Brook University New York

2. Protistan parasite of Mercenaria mercenaria Important fisheries, aquaculture; U.S. East –1 st 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

3. Possible sources of variability In 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

4. 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

5. 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

6. Final cumulative mortality (27 weeks) % mortality Seed type Adjusted by amount of control mortality % mortality Seed type

7. Isolate: NY-1 Isolate: NY-2 Isolate: MA-1 Percent prevalence of infection by isolate. Low High Intermediate

8. 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 -5 Simultaneous interaction of all three variables: NS Two way interactions: S SSeed Type:S*Isolate: G-test of independence for 2 way tables Independence of Seed type NSMA-1: FL ↑S NY-1: VA ↑S NY-2: S Independence of Isolate S**FL:MA:SNY:SVA: **High NY-1; p<10 -4 Lack of prevalence in MA-1 treatment

9. 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 IsolateHard clam type

10. QPX Transmission: Field approach Aim: Infective pressure and dynamics in environment Compare the susceptibility of different clam strains Investigate early stages of the disease Raritan Bay Flanders Bay Southold Bay Northwest 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/site 500 seed/bag QPX prevalence and clam mortality

11. QPX present only in mantle QPX present in ~85% pallial organs N=60 N=120 N=48

12. 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

13. 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

14. 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)

15. 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

16. 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

17. Questions?

18. Thanks for Listening! See you at the…

19. Temperature (ºC) Percent growth (%) Factors affecting QPX growth In vitro growth of QPX cultures: optimal around 20-23° C (different for different isolates), in agreement with QPX prevalence in the field

20. MA-1 N=1 NY8BC7 N=27 NY20AC5 N=6 MA-1 N=5 NY8BC7 N=4 NY20AC5 N=8 MA-1 N=10 NY8BC7 N=13 NY20AC5 N=9 MA-1 N=3 NY8BC7 N=25 NY20AC5 N=10

21. 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 inoculate treatment the clam type and infection 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 more attributed to the isolate of QPX that was injected into the clam P values Interaction term: T-1T-2Moribund Three way 0.110.230.33 SeedType X Inoculate 0.452.52 10 -9 6.04 10 -5 SeedType X InfectionStatus 0.020.010.51 Inoculate X InfectionStatus 2.28 10 -8 0.024.25 10 -6 Factor term: SeedType 0.134.13 10 -11 0.0001 Inoculate 1.75 10 ^-6 6.54 10 -11 1.66 10 -10 InfectionStatus 5.60 10 -9 0.00061.12 10 -5 Complete Independence 5.20 10 -7 3.71 10 -12 9.75 10 -10

22. T-1T-2 Moribund Inoculate PMajor2 nd PMajor2 nd PMajor2 nd MA-1NSlow %’s NSlow %’s NS= 6-32% NY8BC7NS =21-45% 0.0344FL, > NS= 45-55% NY20AC50.0188VA, > NSlow %’s NS= 25-35% T-1T-2Moribund Seed typePMajor2 nd PMajor2 nd PMajor2 nd MA0.0213A, <B, >NS< = 21-45% NY0.0038A, 0 NS< 0.0002A, <B, > VA0.0043A, <C, >NS= 17-24% NS= 32-52% FL3.4 10 -5 B, >A, <0.0107A, <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

23. Cytotoxicity of different QPX isolates Hemocyte viability (OD 560 nm) In vitro toxicity varies among QPX isolates

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

25. 1960’s NB 1989 PEI 1990’s MA NY 2002 1976 NJ 1997 VA survey 2003 NY, RI QPX disease; History and Geographic distribution 1990’s NS survey