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)

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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

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Temperature and shell disease

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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.