Download 2_Chisto+Gille+Shiel+Tayl+Tlus_Baird2010

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
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
4 methods
DGGE
LH-PCR
Multitag Pyrosequencing
TFRLP
Lots of bacteria on the shell
Areas within a lobster differ….
Can identify bacteria
A different analysis – same result
Healthy vs diseased:
Distinct differences
Change with increasing disease state
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.
Bacteria change in abundance with disease
Bacteria
Jannaschia
Aquimarina
Cardiobacterium
Cycloclasticus
Leisingera
Hirschia
Leucothrix
Ahrensia
Filomicrobium
Achromobacter
Dokdonia
Delftia
Kangiella
Corynebacterineae
Lactococcus
Hyphomicrobium
Haliscomenobacter
Diseased
0.1725
0.1350
0.0876
0.0200
0.0143
0.0126
0.010
0.0087
0.0086
0.0081
0.0062
0.0044
0.0041
0.0032
0.0030
0.0027
0.0018
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Std. Dev.
0.1213
0.1486
0.084
0.0302
0.0542
0.0254
0.0230
0.0223
0.0267
0.0312
0.0247
0.0315
0.0135
0.0096
0.0208
0.0207
0.0080
Healthy
0.0712
0.0250
0.0836
0.0290
0.0101
0.0020
0.0180
0.0069
0
0.0060
0.0041
0.0011
0.0031
0.0241
0.0041
0
0.0005
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Std. Dev.
0.0601
0.1019
0.0959
0.0621
0.0173
0.0069
0.0281
0.010
0
0.0194
0.0118
0.0045
0.0085
0.0457
0.0180
0
0.0026
* Entries in red indicate that the genus is more abundant in the diseased state.
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 & diseased
shells have different memberships but much overlap
• Change in abundance of species between
healthy shell and shell with disease lesions
• 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
• 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)
What type of bacteria settle
onto lobster shell?
?
Bulk of microbiome the
Compare bacteria in Healthy
same
What type of bacteria first
vsbetween
lesionedhealthy
shell
attack the lobster shell?
and diseased lobsters,
but are some differences.
What type of bacteria are present
as the lesion worsens?
Why do only some
lobsters get
shell disease?
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
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
– Aquimarina ‘homaria’
(R / L side)
– -proteobacterium
(R side)
Marinosulfonomonas methylotropha
– Pseudoalteromonas gracilis
(R side)
What type of bacteria settle
onto lobster shell?
What type of bacteria first
attack the lobster shell?
Aquimarina ‘homaria’
Use established community to
understand
“pioneers”
Simple laboratory model
What type of bacteria are present
as the lesion worsens?
Why do only some
lobsters get
shell disease?
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.
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
Increase lesions, decrease growth
15C - 6th molt
40
Length
F1,12=7.9, p < 0.02 r2 = 0.40
%Change
Weight
30
20
10
0
0
10
20
30
S Lesion
40
50
60
Damage and shell disease
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