Microbiology of shell disease – which bacteria are responsible?

1. Microbiology of shell disease – which bacteria are responsible? Dr. Andrei Chistoserdov – Univ. Louisiana Dr. Patrick Gillevet – George Mason Univ. Dr. Jeff Shields – Virginia Inst. Marine Science Dr. Gordon Taylor – Stony Brook University Dr. Michael Tlusty – New England Aquarium And a host of others

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 4 methods DGGE LH-PCR Multitag Pyrosequencing TFRLP

6. Lots of bacteria on the shell

7. Areas within a lobster differ….

8. Can identify bacteria

9. A different analysis – same result

10. Healthy vs diseased: Distinct differences

11. Change with increasing disease state

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. Bacteria change in abundance with disease * Entries in red indicate that the genus is more abundant in the diseased state.

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

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

16. Bacterial communities associated with healthy & diseased shells have different memberships but much overlap

17. Change in abundance of species between healthy shell and shell with disease lesions

18. In Healthy 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

19. In Lesions Members of the normal microflora are displaced Dominant bacteria members of common coastal bacterial groups: -proteobacteria , Firmicutes phyla , Rhodobacteraceae and Rhizobiales. Clostridium sp. was successfully cultured anaerobically suggests anoxic conditions in lesions (as in gangrene)

20. What type of bacteria settle onto lobster shell? ? Bulk of microbiome the same between healthy and diseased lobsters, but are some differences. 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?

21. A laboratory model of shell disease Observe many lobsters Known history (diet and genetics) See initial stages and progression of disease Simplified bacterial community / thermal environment

26. A ‘homaria’ lesion spot Kopriimonas byunsanensis Alphaproteo

27. Lesion has >104 more bacteria than healthy surface

28. Can we intentionally create infections? • Bacteria onto filters - attach to lobsters • Aquimarina ‘homaria’ (R / L side) • a-proteobacterium (R side) Marinosulfonomonas methylotropha • Pseudoalteromonas gracilis (R side)

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

31. Aquimarina ‘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.

32. A ‘homaria’ in other arthropods

33. 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

34. 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

35. 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

36. 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

37. 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

38. Temperature and shell disease

39. Temperature and shell disease

40. Temperature and spots lesion spot

41. Temperature and lesions lesion spot

42. Increase lesions, decrease growth

43. Damage and shell disease

44. Opportunisticpolymicrobial disease Shell disease bacteria exists in environment and on other common arthropods (crabs) Bulk of microbiome the same between healthy and diseased, but minor differences community membership (4 methods) More significant is the change in the relative abundance of members of the normal microflora. In model system increased abundance of Aquimarina ‘homaria’ isassociated with first attack Bacteria attack all lobsters equally but outcome influenced by temperature, molt cycle length, animal status Conclusions