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Immune response to acute bacterial
exposure in the American lobster: Do
the tissues play a role in clearing
bacteria from the hemoylmph?
Alyssa McManaway
Advisor : Dr. Jorgensen
http://www.123rf.com/photo_7980498_american-lobster-homarusamericanus-in-front-of-white-background.html
Content
 Introduction
 Materials and Methods
 Results
 Conclusions
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u=3648795608
The American Lobster
 Homarus americanus
 Lives along the Atlantic Coast of North America
 Migratory animals
 Commercially important
 Open circulatory system
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Comparative Circulatory System Information
 Lobsters have an open circulatory system as compared
to other organisms
 As compared to the closed system of vertebrates
 Vertebrates have a microcirculation
 Our vascular system is completely confined to a tube
network
 Compared to Lobsters-
Lobster Circulatory system

Hemolymph is not confined only in the vessel network

The hemolymph leaves the arterial network and bathes the tissue cells directly before
being collected in the venous network

Material exchanged happens across the tissue cell membranes

The space outside the tube network is called the hemocoel, essentially the
extracellular space

The ventricle is contractive and pumps hemolymph through the circulatory system.

Gills are the site of gas exchange, see all of cardiac output

Gills are immune organs
http://www2.gsu.edu/~bioasx/closeopen.html
http://betournay.wikispaces.com/Cir
culation+and+Gas+ExchangeA
Lobster Immune System
 Two part system –
 Circulating hemocytes – analogs of our white blood cells
 Fixed phagocytes
Circulating Hemocytes
 Circulating hemolymph cells are able to differentiate
self from foreign
 When a pathogen is sensed hemocytes quickly mobilize
to the site of infection
 Hemocytes adhere to the cell surface of the pathogen
(Caiwen and Shields,2007)
Nodule Formation
• In vitro experiment
• Hemocytes become sticky and
clump together to form nodules
• These nodules get stuck in the
gills and perhaps other tissues?
(Martin, et al. 1998)
Fixed Phagocytes
 Phagocytotic cells that are
stationary on tissue membranes
 As hemolymph baths the tissues
these cells phagocytose
pathogenic particles
 Have been found in the
Hepatopancreas/digestive glad
 Hepatopancreas is a digestive
gland– functions as a liver and in
digestion
http://encyclopedia.lubopitko-bg.com
Body_Defenses.html
My Research Question
 Do the tissues of the lobster play a part in
bacterial clearance from the hemolymph?
 If so, which tissues are important?
Materials and Methods
Materials and Methods
 Prep Lobster by drilling 0.5mm holes into the carapace
until the endomembrane was visible using sterile
techniques
 The day prior to the experiment GM Vibrio campbellii
was plated and allowed to grow for 24 hours
 Resistant to two antibiotics – for pure cultures
 On the day of the experiment the bacteria was
suspended in a saline solution until the number of
bacteria was equal to 2x108 Colony forming units
 Allows for bacterial infection, but not a lethal dose
GM Vibrio campbellii
 Naturally occurring ocean bacterium
 Bio-engineered to be resistant to kanamycin and
chloramphenicol
 Expresses GFP
 This allows us to track the bacteria in whole animals
 Free from the masking effects of other bacteria
Hemolymph Sampling
 Taken from the
pericardial sinus around
the heart
 Used as an indicator of
successful bacterial
exposure
Gills
Hegner and Engemann 1968 (from Barth and Broshears,1982)
Tissue Sampling
 After 50 minutes the lobster was sacrificed
 At 60 minutes the heart was removed to stop circulation
 The tissues were removed, homogenized in HEPES
(volume was mass dependent) and a subset of the
homogenate was plated
 Bacterial colonies were counted after 24 hours
Tissues Sampled
Ruppert and Barnes, 1994
Gills
Hegner and Engemann 1968 (from Barth and Broshears,1982)
Herrick, 1909 (From Factor, 1995)
Results
Results
 Antennal Gland, Heart, and Hepatopancreas have
highest number of culturable bacteria
 Determined significance using an ANOVA statistical test
and post 2-sample T-tests
*
*
*
n=6
Discussion and Conclusions
Discussion
 The results of the hepatopancreas were consistent with
previous research
 In vitro studies, with inert particles, not bacteria
 The numbers of culturable bacteria in the heart and the
antennal gland were surprising
 Tiny mass – lots of bacteria
 Possible explanations
 Bacteria may not have been phagocytosed
 Nodules may become trapped in the tissues
Discussion
 See very little culturable bacteria in the gills
 Possible explanations
 Nodules in the gills are not culturable
 Gills lack fixed phagocytes (Martin, et al. 1998)
Future Studies
 Interested in other tissues – tail muscle
 Leg muscle, cheliped muscle
 Using the GM vibrio to observe bacterial cells in the
organs
 Future research with hypoxia
Acknowledgements
 Dr. Jorgensen
 Professor Thibodeaux
 Roanoke College Biology Department and the Thornhill
Endowment at Roanoke College
 Grice Marine Laboratory at the College of Charleston, SC
 Current Jorgensen Lab members animal maintenance and
care
 Jorgensen Lab Seniors: Ben Shapiro, Cullen Truett, and Shana
Hensley
Questions?
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References

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
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
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
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
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
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
Martin, G.G., Kay, J., Poole, D., and C. Poole. (1998). In vitro nodule formation in the ridgeback prawn, Sicyonia ingentis,
and the American lobster, Homarus americanus. Invertebrate Biology 117(2): 155-168.

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DAPTIVE IMMUNITY IN THE CRAYFISH