Download Tlusty Taylor Chistoserdov Gillevet Baird presentation final

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
9th Annual Ronald C. Baird Sea Grant
Science Symposium
What type of bacteria settle
onto lobster shell?
What type of bacteria first
attack the lobster shell?
Compare bacteria in Healthy
Vscommunity
lesioned shell
Use established
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
Compare bacteria of healthy
to 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
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
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.
Bacterial community activity
)
300
250
Monomer release rate (mol mm
-2
-1
h )
(destructive enzyme rates)
200
Peptidase
lesion
Lesion Samples
healthy
shellShell
– diseased lobster
Asymptomatic
healthy shell – healthy lobster
Cellulase
300
Asymptomatic Lobster
200
150
100
100
50
0
1400
1200
0
1
2
3
Lipase
4
Chitinase
8000
1000
6000
800
600
4000
400
2000
200
0
Jun07
0
Aug07
Oct07
Jun08 Jun07
Aug07
Oct07
Jun08
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 first
attack the lobster shell?
?
Bulk of microbiome the
Compare bacteria in Healthy
same
vsbetween
lesionedhealthy
shell
and diseased lobsters.
What type of bacteria are present
as the lesion worsens?
Why do only some
lobsters get
shell disease?
A laboratory model of shell disease
New England Aquarium
M Tlusty, A Metzler
Univ Louisiana
A Chistoserdov, R Quinn
Roger Williams Univ
R Smolowitz
A homaria
Kopriimonas
byunsanensis
Alphaproteo
lesion
spot
A. homaria cells/cm2
1.00E+08
1.00E+07
Wild lobsters
1.00E+06
Diet Induced
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
lesion
healthy cararapace
healthy claw
healthy tail
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 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?
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 arthropods
Species
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.
2.
3.
Buzzards Bay Massachusetts, 11 locations
Around Block Island, Rhode Island,
West Connecticut line to east Narragansett bay, Rhode Island
Water
samples at:
10 ft
20 ft
30 ft
40 ft
50 ft
60 ft
70 ft
80 ft
90 ft
100 ft
MUD
Bottom
samples
Ekman Grab Niskin bottle
SAND
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
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 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
10
9
8
7
6
5
4
3
2
1
0
3 year old lobsters
1 year old lobsters
7
0
62
75
87
% Herring in Diet
100
Shell Disease Severity Index
Shell Disease Severity Index
Diet and shell disease
6
5
4
3
Dead
2
1
0
0
62
75
87
% Herring in Diet
100
Temperature and shell disease
50
Total % Areas with SD
Total % Areas with SD
40
10 degrees
15 degrees
20 degrees
30
20
10
0
0
1
2
3
4
5
6
7
Molt #
140.00
Average # of Days in Molt cycle (molts 1 to 2)
120.00
100.00
80.00
60.00
40.00
20.00
0.00
10 degrees
15 degrees
20 degrees
8
Temperature and shell disease
0.6
% Total Areas/Molt Cycle
% Total Areas with SD Corrected for Molt Cycle
0.5
10 degrees
15 degrees
0.4
20 degrees
0.3
0.2
0.1
0
0
1
2
3
4
Molt #
5
6
7
8
Temperature and spots
lesion
30
spot
% Areas with Spots
20
10
10 degrees
15 degrees
20 degrees
0
0
1
2
3
4
Molt #
5
6
7
8
Temperature and lesions
lesion
spot
30
Lesions
% Areas with Lesions
20
10 degrees
15 degrees
20 degrees
10
0
0
1
2
3
4
Molt #
5
6
7
8
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.