Microbiology of shell disease – which bacteria are responsible?

1. 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. Compare bacteria in Healthy Vs lesioned shell Use established community to understand “pioneers” 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

5. Compare bacteria of healthyto lesioned shell 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

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? ? Bulk of microbiome the same between healthy and diseased lobsters. Compare bacteria in Healthy vs lesioned 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?

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. A homaria lesion spot Kopriimonas byunsanensis Alphaproteo

24. Lesion has >104 more bacteria than healthy surface

25. Can we intentionally create infections? • Bacteria onto filters - attach to lobsters • Aquamarina ‘homaria’ (R / L side) • a-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? Aquamarina ‘homaria’ Use established community to understand “pioneers” What type of bacteria are present as the lesion worsens? Why do only some lobsters get shell disease?

28. Aquamarina ‘homaria’ • 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

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

31. Water sample at 20 ft 5ìm fraction positive = 103/L Sand sample at 74 ft deep positive = 107/g Mud sample at Harbour of refuge 26 ft deep (3 cm deep in core) positive = 103/g Sand sample at 37 ft deep positive = 102/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. 3 year old lobsters Shell Disease Severity Index 1 year old lobsters % Herring in Diet Shell Disease Severity Index Dead % Herring in Diet Diet and shell disease

35. Temperature and shell disease

36. Temperature and shell disease

37. Temperature and spots lesion spot

38. Temperature and lesions lesion spot

39. Damage and shell disease

40. 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. Conclusions