1 / 34

Understanding Shell Disease in Lobsters: Insights from Gross and Histopathological Evaluations

This session discusses the gross and histopathological appearance of shell disease in lobsters, as well as the identification of genes expressed in infected lobsters using qPCR methods. It also explores the characterization of exoskeletal microbial communities and the host immune response associated with shell disease.

garciarichard
Download Presentation

Understanding Shell Disease in Lobsters: Insights from Gross and Histopathological Evaluations

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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

  2. Histopathological Lesions Catagories Descriptions of ESD The “100” findings Juvenile lobsters exposed to bacteria at different temperatures Classification of shell diseases in lobsters

  3. NORMAL LOBSTER CUTICLE IN STAGE C4/Do

  4. 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

  5. STAGE 2 MODERATELY DEEP EROSIONS INTO THE CALCIFIED ENDOCUTICLE ***CUTICULAR PILLAR FORMATION*** MELANIZATION LOCALIZED PROLIFERATION OF INFLAMMATORY CUTICLE VARIABLE HYPERPLASIA OF CUTICULAR EPITHELIUM

  6. AT THE LEADING EDGE ARE BACTERIA Aquimarina homaria

  7. “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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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?

  13. An Apatite for Lobster Apatite and Calcite: Resolution of mineral location in lobster cuticle and a potential cause for weakened shells in lobsters

  14. 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

  15. Electron Microprobe Images of Lobster Canals … and Trabeculae: Bone Trabeculae: Cuticle Trabeculae:

  16. Raman Spectroscopy detects Chemical Bonds Canals are well protected from dissolution. Matrix of Raman Spectra Discrimination among Spectra Canal Light Image Phosphate Wall Calcite Ring Canal Contents

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  21. 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)

  22. Arginine Kinase in Muscle Tissue Helps to regulate cellular energy (ATP) stores. Expression severely reduced in muscle of symptomatic lobsters.

  23. EcR + EcRE Ecdysteroid Receptor in Hepatopancreas Tissue Elevated EcR in hepatopancreas of symptomatic lobsters during intermolt RXR “CYP” Enzymes 20-OHE Protein synthesis Molting E

  24. EcR + EcRE CYP Expression Reduced expression of several CYP enzymes in hepatopancreas of symptomatic lobsters RXR “CYP” Enzymes 20-OHE E Disease stage

  25. 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

  26. 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.

  27. 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.

  28. Lobsters from ELIS (Diseased vs. Healthy) A comparison between healthy lobsters from ELIS vs either lobsters from WLIS or Maine

  29. 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

  30. 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.

  31. 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.

  32. 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.

  33. 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

More Related