1. 9th Annual Ronald C. Baird Sea Grant Science Symposium Microbiology of shell disease – which bacteria are responsible? Dr. Gordon Taylor –Stony Brook University Dr. Andrei Chistoserdov – Univ. Louisiana Dr. Patrick Gillevet – George Mason Univ. Dr. Michael Tlusty – New England Aquarium

4. What type of bacteria settle onto lobster shell? What type of bacteria first attack the lobster shell? What type of bacteria are present as the lesion worsens? Why do only some lobsters get shell disease? Compare bacteria in Healthy Vs lesioned shell Use established community to understand “pioneers” What drives this Initial infection

5. Stony Brook Univ. – G. Taylor, B. Allam, A. McElroy, co-PIs; S. Bell, Sea Grant Scholar, and T. Barrett, undergrad George Mason University C Ajuzie, M Sikaroodi, N Meres, P Gillevet Virginia Institute of Marine Sciences Jeff Shields et al Compare bacteria of healthy to lesioned shell

6. Three similar but different methods

7. Lots of bacteria on the shell

8. Areas within a lobster differ….

9. Healthy vs diseased: No distinct differences

10. Can identify bacteria

11. A different analysis – same result

12. Changes in abundance Peak 217bp (AluI digest) in lesion samples represented 4% of the total community profile and much less than 1% in healthy shell samples. lesion sample gene fragment of pathogen or opportunistic species?? healthy shell sample same fragment

13. Bacterial community activity (destructive enzyme rates) lesion healthy shell – diseased lobster healthy shell – healthy lobster

14. Bacteria of healthy vs lesioned shell Genetic signatures of bacteria on shell span multiple major taxonomic groups, potentially comprised of 100’s of species

15. Bacterial communities associated with healthy and diseased shells appear to have similar memberships based on “fingerprinting” technique (TRFLP)

16. Four “species” (restriction fragments) were clearly more abundant in disease lesions compared to healthy shell

17. Community “Fingerprinting” Results In Healthy Shells (n = 84 samples) – 6 peaks more common indicating some members of the normal microflora are displaced from diseased lobster shells. – Numerous potential taxonomic associations, but dominated by members of  - and  -proteobacteria rather than  -proteobacteria as seen in lesions. – No viable bacterial cultures under anaerobic culture conditions

18. Community “Fingerprinting” Results In Lesions (n = 33 samples) – Dominant TRFLP peaks (15-25% of total area) potentially belong to members of common coastal bacterial groups:  -proteobacteria and Firmicutes phyla as well as Rhodobacteraceae and Rhizobiales. – One potential match to Clostridium species (AluI 217bp ) suggests anoxic conditions in lesions (as in gangrene) – Clostridium sp. was successfully cultured anaerobically

19. What type of bacteria settle onto lobster shell? What type of bacteria first attack the lobster shell? What type of bacteria are present as the lesion worsens? Why do only some lobsters get shell disease? Compare bacteria in Healthy vs lesioned shell Bulk of microbiome the same between healthy and diseased lobsters. ?

20. A laboratory model of shell disease New England Aquarium M Tlusty, A Metzler Univ Louisiana A Chistoserdov, R Quinn Roger Williams Univ R Smolowitz

23. Kopriimonas byunsanensis Alphaproteo spot lesion A homaria

24. Lesion has >10 4 more bacteria than healthy surface

25. Can we intentionally create infections? Bacteria onto filters - attach to lobsters – Aquamarina ‘homaria’ (R / L side) –  -proteobacter(R side) – Pseudoalteromonas gracilis (R side)

27. What type of bacteria settle onto lobster shell? What type of bacteria first attack the lobster shell? What type of bacteria are present as the lesion worsens? Why do only some lobsters get shell disease? Use established community to understand “pioneers” Aquamarina ‘homaria’

28. Based on 16S rRNA and phospholipid fatty acid composition is a species different from but closely related to A. muelleri. Not commonly found in the environment. Aquimarina muelleri is found in sediments, associated with algae and marine invertebrates. Apart from Arthropods, was detected only in a sea hatchery in Canada.

29. “A homaria” in other arthropods SpeciesLesionsHealthy Carapace Lobster1720 Spider Crab108 Green Crab73 Jonah Crab98 Horseshoe Crab 43

30. Environmental Sampling Samples taken from three different trips 1.Buzzards Bay Massachusetts, 11 locations 2.Around Block Island, Rhode Island, 3.West Connecticut line to east Narragansett bay, Rhode Island MUDSAND Water samples at: 10 ft 20 ft 30 ft 40 ft 50 ft 60 ft 70 ft 80 ft 90 ft 100 ft Ekman Grab Niskin bottle Bottom samples

31. Sand sample at 74 ft deep positive = 10 7 /g Water sample at 20 ft 5ìm fraction positive = 10 3 /L Mud sample at Harbour of refuge 26 ft deep (3 cm deep in core) positive = 10 3 /g Sand sample at 37 ft deep positive = 10 2 /g

32. Environmental Sampling Summary A. ‘homaria’ - detected on other invertebrates A. ‘homaria’ - also detected on lobster bait (skate and haddock) A. ‘homaria’ is not a common marine bacterium Appears to be present in more off shore sand sediments Unusual distribution in New England Aquarium

33. What type of bacteria settle onto lobster shell? What type of bacteria first attack the lobster shell? What type of bacteria are present as the lesion worsens? Why do only some lobsters get shell disease? What drives this Initial infection

34. Diet and shell disease % Herring in Diet Shell Disease Severity Index 3 year old lobsters 1 year old lobsters % Herring in Diet Shell Disease Severity Index Dead

35. Temperature and shell disease

37. Temperature and spots spot lesion

38. Temperature and lesions spot lesion

39. Damage and shell disease

40. Conclusions Pioneer is A. ‘homaria’ - first bacteria through shell – Influenced by temperature, molt cycle length, animal status Likely natural reservoir of A. ‘homaria’ is various arthropods (crabs) Recently evolved to infect compromised lobsters in southern New England (host susceptibity) Overall bacterial community memberships in diseased and healthy shells were not grossly different according to three different methods. – ESD appears to induce subtle changes in the relative abundance of members of the normal microflora.