Transcript 3_Tarr+Smol+Kunk+McElr

Discussed in this session.
 The gross and histopathological appearance of
shell disease – R. Smolowitz
 Apatite and calcite: resolution of mineral location
in lobster cuticle – J. Kunkel
 Identification of genes expressed in infected
lobsters with qPCR methods – A. Tarrant and T.
Versylcke
 Characterization of ther exoskeletal microbial
communities and host immune response
associated with ESD – B. Allam, A. Dove, A.
McElroy, G. Taylor, M. Fast
Histopathological Lesions
Catagories
 Descriptions of ESD
 The “100” findings
 Juvenile lobsters exposed to bacteria at
different temperatures
 Classification of shell diseases in lobsters
NORMAL LOBSTER CUTICLE IN STAGE C4/Do
EPIZOOTIC SHELL DISEASE
HISTOPATHOLOGY OF CARAPACE LESIONS
 4 STAGES
 STAGE 1
 SHALLOW EPICUTICULAR AND EXOCUTICULAR
LESIONS
 STAGE 2
 MODERATELY DEEP EROSIONS INTO THE
CALCIFIED ENDOCUTICLE SHOW
 STAGE 3
 SEVERE EROSIONS INTO UNCALCIFIED
ENDOCUTICLE
 STAGE 4
 ULCERATION
STAGE 2

MODERATELY DEEP EROSIONS
INTO THE CALCIFIED ENDOCUTICLE
 ***CUTICULAR PILLAR
FORMATION***
 MELANIZATION
 LOCALIZED PROLIFERATION
OF INFLAMMATORY CUTICLE
 VARIABLE HYPERPLASIA OF
CUTICULAR EPITHELIUM
AT THE LEADING EDGE ARE BACTERIA
Aquimarina homaria
“THE 100”
 MOST TISSUE SUBMITTED
TAKEN FROM THE EDGE
OF THE CARAPACE OR
FROM ABDOMENAL
SEGMENTS (NOT DORSAL
MIDLINE)
 ERODED EXOCUTICLE IN
CA++ CARBONATE ZONES
BETWEEN PHOSPHATE
TRABECULAE
 IDENTIFIED BY DR.
KUNKEL
HISTOPATHOLOGICAL EVALUATION OF
CARAPACE FROM INFECTION STUDIES
(Tlusty et. al.)
Review of experimental methods:
Lobsters held at 3 temperatures (10, 15 and 20º C)
Scraped on lateral sides of the carapace
Bacterial applied via filter paper to the scraped
carapace
Left – A. homaria only
Right – Mixture of A. homaria and 2 other bacteria
HISTOPATHOLOGICAL EVALUATION OF
CARAPACE FROM INFECTION STUDIES
 RESULTS:
 No real differences between A. homaria only and mixed
bacterial infections containing A. homaria
 On the left and right side of the same animals
 Between animals at the same temperatures
 Between animals at different temperatures
 Note: A. homaria was identified in the other lesions on the
animals and in the systems.
HISTOLOGICAL EVALUATION OF CARAPACE FROM
INFECTION STUDIES
 PILLAR-LIKE
REMNANTS WERE
NOTED RARELY
 15 AND 20 °c
INFECTED
ANIMALS
 ATTACHED FILTER
PAPER MIGHT HAVE
DECREASE PILLAR
LIKE MATERIAL
HISTOPATHOLOGICAL EVALUATION OF
CARAPACE FROM INFECTION STUDIES
 RESULTS:
 Interestingly, the inflammatory membrane production appeared
to be most pronounced at the 15° C temperature?
 IN BOTH - Control (scraped) and Infected (scraped and
infected) animals!
 Did not appear to be related to whether animal was in
carapace formation stage C4/D0 but rather temperature.
Summary
Histopathology
 Other causes of shell disease must be
distinguished from ESD with history, gross and
microscopic evaluation of lesions.
 Cuticular pillars are common in ESD vs. other
types of shell disease in American lobsters
 Shell disease can be induced in laboratory
 What does lack of an inflammatory membrane
in experimental at high and low temperature
mean?
An Apatite for
Lobster
Apatite and Calcite: Resolution of mineral location in lobster cuticle
and a potential cause for weakened shells in lobsters
How the Kunkel Lab Studies
Lobster? (two types of tissue preparations)
Fixed material:
•Experimental Design
•Excise cuticle …
•Fixation …
•Physical methods …
•Analysis …
Live material:
•Experimental Design
•Treatments …
•Non-invasive probe
•Analysis
Electron Microprobe Images of Lobster Canals
… and Trabeculae:
Bone
Trabeculae:
Cuticle Trabeculae:
Raman Spectroscopy detects Chemical Bonds
Canals are well protected from dissolution.
Canal Light Image
Canal Contents
Matrix of Raman Spectra
Calcite Ring
Discrimination among Spectra
Phosphate Wall
Oxygen Electrode follows O2 Flux into
Melanizing Lesions.
Oxygen flux is maximum at 2.63 hours post lesion induction.
Primary response is increased CaCO dissolution
Secondary response is a proPhenoloxidase activation
Measuring ion and oxygen flux through the cuticle.
Model Showing Bone-Canals
and -Trabeculae & Calcite
Ocean bottom tends to be more acidic. Increased destruction of the calcite layer
from ocean acidification may predispose to shell disease.
A.Tarrant and T. Versylcke Research
Tools and resources that were developed
•
Created 4 subtractive gene libraries from symptomatic and
asymptomatic lobsters, generated ~400 mRNA sequences
• Developed qPCR assays to measure expression of 28 genes
including hormone receptors, p450 enzymes, and immuneresponsive genes.
Resource and information sharing
•
identified ~ 400 mRNA sequences
•submitted to the public database (GenBank).
• Publication resulting from the work - Gene expression patterns
and assay conditions described in : Tarrant AM, Stegeman JJ,
Verslycke T. (In Press) Altered gene expression associated with
epizootic shell disease in the American lobster, Homarus
americanus. Fish and Shellfish Immunology.
Data highlights from the “100 lobsters” project:
Analyzed 36 female lobsters:
12 asymptomatic, 12 stage I, 12 stage 2-3.
Most (30/36) intermolt
Muscle (Arginine Kinase, Hsp70, Hsp90, EcR)
Hepatopancreas (Cyps, Retinoid x Receptor (RxR),
EcR, Hemocyanin)
Ovary (a2-macroglobulin, RXR, EcR)
Hemocytes (macroglobulin, crustins, chitinases)
Arginine Kinase in Muscle Tissue
Helps to regulate cellular energy (ATP) stores.
Expression severely reduced in muscle of
symptomatic lobsters.
3.00E+09
Mean Molecule Number
2.50E+09
2.00E+09
1.50E+09
1.00E+09
5.00E+08
0.00E+00
0
1
Stage
2
3
3.00E+04
2.50E+04
Ecdysteroid Receptor in
Hepatopancreas Tissue
2.00E+04
1.50E+04
Elevated EcR in
hepatopancreas of
symptomatic lobsters
during intermolt
1.00E+04
5.00E+03
0.00E+00
0
1
2
3
EcR RXR
“CYP”
Enzymes
+
EcRE
20-OHE
E
Protein synthesis
Molting
"Shade"
CYP Expression
Reduced expression of several CYP
enzymes in hepatopancreas of
symptomatic lobsters
"Cyp330"
"Locust Cyp"
EcR RXR
“CYP”
Enzymes
+
EcRE
20-OHE
E
0
1
2
Disease stage
3
What do we know now?
•
Arginine kinase
• expression is low in muscle of symptomatic lobster. Diseased
lobsters may be energetically compromised.
• Ecdysteroid Receptor (EcR)
• During intermolt, symptomatic female lobsters showed
elevated ecdysteroid receptor expression. Along with
previous observations of elevated ecdysone (Laufer), this
indicates that shell disease is associated with disruption of
hormonal signaling.
•Cytochrome P450 (CYP) Expression--Reduced
-Decreased steroid synthesis?
-Decreased xenobiotic metabolism?
-Disruption of normal hormonal balance in shell diseased animals
•
Research by B. Allam, A. Dove, A.
McElroy, G. Taylor and M. Fast
Characterization of the exoskeletal
microbial communities and
HOST IMMUNE RESPONSE
associated with epizootic shell disease.
Sampling took advantage of
a large difference in
disease prevalence
between ELIS and WLIS
lobsters. Lobsters from
Maine used as a reference
population.
In addition to microbial
analyses (as discussed
previously), we focused
on differences in
internal and external
defense parameters
between the three
populations.
Lobsters from ELIS
(Diseased vs. Healthy)
A comparison between
healthy lobsters from ELIS
vs either lobsters from WLIS
or Maine
Principle component analysis
(PCA) of all lobsters sampled
indicates that immune
responses (phagocytosis,
phenoloxidase, antibacterial
activity) are negatively
correlated with measures of
disease status (MAXcfu,
Disease Code)
Discriminant analysis of
only healthy lobsters
demonstrates that ELIS
lobsters are significantly
different from both WLIS
and Maine lobsters
Defense parameters of the shell:
 Lobster shell (from all locations) represents a constitutive first line of
biochemical defense (not just physical) against bacterial challenge.
Extracts of shell show antimicrobial activity against a number of
gram negative and positive bacteria in growth challenge studies
 Characterization of this activity indicates it is:
 small - likely some kind of peptide <10K in size
 heat stabile - boiling has no effect, ashing at 450oC for several
hours only diminished activity by half
 There appears to be no significant differences in antimicrobial
activity in shell extracts from lobsters as a function of sampling
location or disease status.
What we know now:
Diseased lobsters show depressed immune
capacity as compared to healthy (asymptomatic)
individuals.
 Healthy lobsters from ELIS, show depressed
immune capacity as compared to lobsters form
WLIS or Maine indicating that the ELIS population
may be compromised in some way.
Lobster carapaces have antimicrobial activity.
GENERAL CONCLUSIONS
 Epizootic Shell Disease is differentiated from other types




by the number of animals affected and by the histological
appearance
Other crustaceans in the northeast are not affected by ESD
Environmental changes or specific infectious agents may
preferentially destroy the calcium carbonate laden areas of
the shell.
Molecular evidence demonstrates that ESD affected
lobsters have reduced ability to process xenobiotics
(reduced P450s), reduced steroid synthesis with increased
numbers of molting receptors leading to disruption of
normal metabolic and molting mechanisms.
Immune capacity
 Diseased lobsters show depressed immune capacity
 Healthy lobsters from ELIS, show depressed immune capacity as
compared to lobsters form WLIS or Maine.
 Lobster carapaces have antimicrobial activity.
FUTURE STUDIES





Effects of temperature on carapace formation
Effects of pH on carapace dissolution
Function of individual CYPs and consequences of
altered expression
Effects of disease on lobster metabolism (energy) and
reproduction
Characterization of properties and factors influencing
defense-related factors in lobster shell disease