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Discussed in this session. The gross and histopathological The gross and histopathological
appearance of shell disease – R. appearance of shell disease – R. Smolowitz 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 EPIZOOTIC SHELL DISEASE HISTOPATHOLOGY OF CARAPACE LESIONSHISTOPATHOLOGY OF CARAPACE LESIONS
4 STAGES 4 STAGES STAGE 1STAGE 1
SHALLOW EPICUTICULAR AND SHALLOW EPICUTICULAR AND EXOCUTICULAR LESIONSEXOCUTICULAR LESIONS
STAGE 2STAGE 2 MODERATELY DEEP EROSIONS INTO THE MODERATELY DEEP EROSIONS INTO THE
CALCIFIED ENDOCUTICLE SHOWCALCIFIED ENDOCUTICLE SHOW
STAGE 3STAGE 3 SEVERE EROSIONS INTO UNCALCIFIED SEVERE EROSIONS INTO UNCALCIFIED
ENDOCUTICLEENDOCUTICLE
STAGE 4STAGE 4 ULCERATIONULCERATION
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 BACTERIAAquimarina 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 carapaceBacterial applied via filter paper to the scraped carapace
Left – A. homaria onlyRight – 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
BoneTrabeculae: Cuticle Trabeculae:
… and Trabeculae:
Raman Spectroscopy detects Chemical Bonds Canals are well protected from dissolution.
Canal Contents Calcite Ring Phosphate Wall
Matrix of Raman Spectra Discrimination among SpectraCanal Light Image
Oxygen Electrode follows O2 Flux into Melanizing Lesions.
Measuring ion and oxygen flux through the cuticle.
Oxygen flux is maximum at 2.63 hours post lesion induction.Primary response is increased CaCO dissolutionSecondary response is a proPhenoloxidase activation
Model Showing Bone-Canals and -Trabeculae
& CalciteOcean 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 immune-responsive 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.
RXREcR+
20-OHE
EcRE
E
“CYP” Enzymes
Protein synthesisMolting
Ecdysteroid Receptor in Hepatopancreas Tissue
Elevated EcR in hepatopancreas of symptomatic lobsters during intermolt
RXREcR+
20-OHE
EcRE
E
“CYP” Enzymes
Disease stage
CYP ExpressionReduced expression of several CYP enzymes in hepatopancreas of symptomatic lobsters
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