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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. LouisianaDr. Patrick Gillevet – George Mason Univ.
Dr. Michael Tlusty – New England Aquarium
What type of bacteria settle onto lobster shell?
What type of bacteria firstattack the lobster shell?
What type of bacteria are presentas the lesion worsens?
Why do only some lobsters get
shell disease?
Compare bacteria in HealthyVs lesioned shellUse established community to
understand “pioneers”
What drives this Initial infection
Stony Brook Univ. – G. Taylor, B. Allam, A. McElroy, co-PIs;
S. Bell, Sea Grant Scholar, and T. Barrett, undergrad
George Mason UniversityC Ajuzie, M Sikaroodi, N Meres,
P Gillevet
Virginia Institute of Marine Sciences Jeff Shields et al
Compare bacteria of healthyto lesioned shell
Three similar but different methods
Lots of bacteria on the shell
Areas within a lobster differ….
Healthy vs diseased: No distinct differences
Can identify bacteria
A different analysis – same result
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 samplegene fragment of pathogen or opportunistic species??
healthy shell sample
same fragment
Bacterial community activity(destructive enzyme rates)
1 2 3 4
0
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100
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300
Lesion SamplesAsymptomatic ShellAsymptomatic Lobster
0
200
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600
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1400
Mon
omer
rele
ase
rate
( m
ol m
m-2
h-1
) )
Peptidase
Lipase
Jun07 Aug07 Oct07 Jun08
0
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Cellulase
Chitinase
Jun07 Aug07 Oct07 Jun08
lesionhealthy shell – diseased lobsterhealthy shell – healthy lobster
Bacteria of healthy vs lesioned shell• Genetic signatures of bacteria
on shell span multiple major taxonomic groups, potentially comprised of 100’s of species
• Bacterial communities associated with healthy and diseased shells appear to have similar memberships based on “fingerprinting” technique (TRFLP)
• Four “species” (restriction fragments) were clearly more abundant in disease lesions compared to healthy shell
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
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
What type of bacteria settle onto lobster shell?
What type of bacteria firstattack the lobster shell?
What type of bacteria are presentas the lesion worsens?
Why do only some lobsters get
shell disease?
Compare bacteria in Healthyvs lesioned shell
Bulk of microbiome the
same between healthy
and diseased lobsters.?
A laboratory model of shell diseaseNew England Aquarium
M Tlusty, A Metzler
Univ Louisiana A Chistoserdov, R Quinn
Roger Williams UnivR Smolowitz
Kopriimonas byunsanensisAlphaproteo
spotlesion
A homaria
Lesion has >104 more bacteria than healthy surface
Can we intentionally create infections?
• Bacteria onto filters - attach to lobsters– Aquamarina ‘homaria’ (R / L side)– -proteobacter (R side)– Pseudoalteromonas gracilis (R side)
What type of bacteria settle onto lobster shell?
What type of bacteria firstattack the lobster shell?
What type of bacteria are presentas the lesion worsens?
Why do only some lobsters get
shell disease?
Use established community to understand “pioneers”
Aquamarina ‘homaria’
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.
“A homaria” in other arthropodsSpecies Lesions Healthy
Carapace
Lobster 17 20
Spider Crab 10 8
Green Crab 7 3
Jonah Crab 9 8
Horseshoe Crab
4 3
Environmental Sampling• Samples taken from three different trips
1. Buzzards Bay Massachusetts, 11 locations2. Around Block Island, Rhode Island, 3. West Connecticut line to east Narragansett bay, Rhode Island
MUD SAND
Water samples at:
10 ft20 ft30 ft40 ft50 ft60 ft70 ft80 ft90 ft100 ft
Ekman Grab Niskin bottle
Bottomsamples
Sand sample at 74 ft deep positive = 107/g
Water sample at 20 ft 5ìm fraction positive = 103/L
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
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
What type of bacteria settle onto lobster shell?
What type of bacteria firstattack the lobster shell?
What type of bacteria are presentas the lesion worsens?
Why do only some lobsters get
shell disease?
What drives this Initial infection
Diet and shell disease
0123456789
10
0 62 75 87 100
% Herring in Diet
She
ll D
isea
se S
ever
ity I
ndex
3 year old lobsters
0
1
2
3
4
5
6
7
0 62 75 87 100
1 year old lobsters
% Herring in Diet
She
ll D
isea
se S
ever
ity I
ndex
Dead
Temperature and shell disease
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
10 degrees 15 degrees 20 degrees
Average # of Days in Molt cycle (molts 1 to 2)
Temperature and shell disease
Temperature and spots
spotlesion
Temperature and lesions
spotlesion
Damage and shell disease
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.