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