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. What type of bacteria settle
onto lobster shell?
Compare bacteria in Healthy
Vs lesioned shell
What type of bacteria first
attack the lobster shell?
Use established community to
understand “pioneers”
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 healthy to 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
lesion sample
gene fragment of pathogen or opportunistic species??
healthy shell sample
same fragment
Peak 217bp (AluI digest) in lesion samples represented 4% of the total community profile and much less than 1% in healthy shell samples.

13. Bacteria change in abundance with disease
Diseased
Std. Dev.
Healthy
Jannaschia
0.1725 +
0.1213
0.0712
0.0601
Aquimarina
0.1350
0.1486
0.0250
0.1019
Cardiobacterium
0.0876
0.084
0.0836
0.0959
Cycloclasticus
0.0200
0.0302
0.0290
0.0621
Leisingera
0.0143
0.0542
0.0101
0.0173
Hirschia
0.0126
0.0254
0.0020
0.0069
Leucothrix
0.010
0.0230
0.0180
0.0281
Ahrensia
0.0087
0.0223
Filomicrobium
0.0086
0.0267
Achromobacter
0.0081
0.0312
0.0060
0.0194
Dokdonia
0.0062
0.0247
0.0041
0.0118
Delftia
0.0044
0.0315
0.0011
0.0045
Kangiella
0.0135
0.0031
0.0085
Corynebacterineae
0.0032
0.0096
0.0241
0.0457
Lactococcus
0.0030
0.0208
Hyphomicrobium
0.0027
0.0207
Haliscomenobacter
0.0018
0.0080
0.0005
0.0026
* 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
In lesion samples, two enzymes (peptidase and cellulase) significantly more active than in healthy shell.
Lipase activity surprisingly high in healthy lobsters in Jun’08
Chitinase activity much higher than other enzymes, but not significantly higher in lesions.

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. Members of the normal microflora are displaced
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

23. Internal spots?

24. Claws – many tiny spots and unusual (internal?) spot

26. Kopriimonas byunsanensis
Alphaproteo
A ‘homaria’
spot
lesion

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)

29. Sides look similar 29

30. Aquimarina ‘homaria’ 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
Species
Lesions
Healthy Carapace
Lobster 17 20
Spider Crab 10 8
Green Crab 7 3
Jonah Crab 9
Horseshoe Crab 4

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

38. Temperature and shell disease

39. Temperature and shell disease

40. Temperature and spots
spot
lesion

41. Temperature and lesions
spot
lesion

42. Increase lesions, decrease growth

43. Damage and shell disease

44. Conclusions Opportunistic polymicrobial 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’ is associated with first attack
Bacteria attack all lobsters equally but outcome influenced by temperature, molt cycle length, animal status