Understanding Understanding Coral HistologyCoral Histology- How & Why -- How & Why -

Ha-Rim Cha

Division of Invertebrate ZoologyThe Natural History Museum of University

of Kansas and Biodiversity Research Center

Coral Slide Reading Workshop

Mote Marine Laboratory, Summerland Key, FL

To learn histological techniques & slide reading skills for scleractinians

Phylogeny of Phylogeny of CorallimorphariaCorallimorpharia

Cylindrical bodyCylindrical body

Family Family Sideractiidae Sideractiidae

Family Family CorallimorphidaCorallimorphidaee

Plate-like bodyPlate-like body

Family RicordeidaeFamily Ricordeidae

Family Family DiscosomatidaeDiscosomatidae

Questions about relationships within the order

Vincent B. Hargreaves

Vincent B. Hargreaves

Questions about relationships between Questions about relationships between Corallimorpharia and two closest orders Actiniaria Corallimorpharia and two closest orders Actiniaria (sea anemones) and Scleractinia (stony corals)(sea anemones) and Scleractinia (stony corals)

↑ Stony coral Corallimorpharia ↑ ↑ Sea anemones

Phylogeny of Phylogeny of CorallimorphariaCorallimorpharia

Characters to be Characters to be examinedexamined

Scott R. Santos

Calcareous exoskeleton

Polyp shape / size

Tentacle shape / number / arrangement

Type of mesenteries

Mesentery number / arrangement

Development of musculature

Nematocyst composition

Life style – solitary or colonial

Habitat

Endosymbiotic relationships

Characters known about Characters known about Coral AnatomyCoral Anatomy

Calcareous exoskeleton

Polyp shape / size

Tentacle shape / number / arrangement

Type of mesenteries

Mesentery number / arrangement

Development of musculature

Nematocyst composition

Life style – solitary or colonial

Habitat

Endosymbiotic relationships

Characters used for Characters used for Coral Taxonomy & Coral Taxonomy &

PhylogenyPhylogeny Taxonomy of Taxonomy of

scleractinian scleractinian corals is based on corals is based on their their skeletal structureskeletal structure

Copyright @ Brin Edwards

Taxonomy of scleractinian coralsTaxonomy of scleractinian corals

Genus Genus FungiaFungia

Genus Genus TubastreaTubastrea

Characters used for Characters used for Coral Taxonomy & Coral Taxonomy &

PhylogenyPhylogeny

Characters used for Characters used for Coral Taxonomy & Coral Taxonomy &

PhylogenyPhylogeny Order level phylogeny of HexacoralliaOrder level phylogeny of Hexacorallia

?Scleractinia

ActiniariaCorallimorpharia

PhylogenyPhylogeny

Order level phylogeny of HexacoralliaOrder level phylogeny of Hexacoralliaa. Chen et al. (1995): 28S rRNA

d. Daly et al. (2003): Morphology, 16S, 18S and 28Sc. France et al. (1996): 16S rRNA

b. Fautin & Lowenstein (1994): radioimmunoassay

Microscopic Polyp Microscopic Polyp AnatomyAnatomy

Additional characters on coral morphologyAdditional characters on coral morphology

An independent character set to compare An independent character set to compare

with published molecular data with published molecular data

HistologyHistology

Histology (= Microscopic anatomy) Histology (= Microscopic anatomy) is…is…

the study of cells, tissues, organs, the study of cells, tissues, organs, and organ systems of plants and and organ systems of plants and animalsanimals

HistologyHistology

Histology (= Microscopic anatomy) is…Histology (= Microscopic anatomy) is…

the study of cells, tissues, organs, and the study of cells, tissues, organs, and organ systems of plants and animalsorgan systems of plants and animals

Structure & Composition of an organism Structure & Composition of an organism or or

a part reflects its key functions a part reflects its key functions

(e.g. respiration, metabolism, (e.g. respiration, metabolism, reproduction, movement)reproduction, movement)

Coral HistologyCoral Histology To support studies on ecology, To support studies on ecology,

physiology, reproduction, biochemistry, physiology, reproduction, biochemistry, immunology, embryology, and systematicsimmunology, embryology, and systematics

Histopathology -- Study on coral diseases Histopathology -- Study on coral diseases (e.g. white-band disease, black-band (e.g. white-band disease, black-band disease, red-band disease) or bleaching disease, red-band disease) or bleaching eventevent

Bleaching & Coral Bleaching & Coral DiseaseDisease

Black-Band Disease

( photos by E. Peters)

Yellow-Band Disease

Bleaching

ALIVE!

DEAD

Procedure for coral Procedure for coral histologyhistology

CollectiCollectingng

FixationFixation

Decalcifying and Decalcifying and TrimmingTrimming

EmbeddiEmbeddingng

SectioningSectioning

StainingStaining

Specimen CollectionSpecimen Collection

Using a cold chisel and a coring Using a cold chisel and a coring device (for a large colony)device (for a large colony)

Relax the collected specimensRelax the collected specimens Obtaining permission prior to Obtaining permission prior to

conducting the studyconducting the study Reporting export and import of Reporting export and import of

collected materials to the United collected materials to the United States Fish and Wildlife Service is States Fish and Wildlife Service is requiredrequired

FixationFixation

To preserve the structure and To preserve the structure and composition of the tissuecomposition of the tissue

Post-mortem effects are usually Post-mortem effects are usually prevented by denaturing and prevented by denaturing and precipitating protein, or by cross-precipitating protein, or by cross-linking the proteins with aldehyde linking the proteins with aldehyde and/or other chemicalsand/or other chemicals

FixationFixation The choice of fixative will depend on the The choice of fixative will depend on the

embedding medium to be used.embedding medium to be used.

for light microscopy, paraffin embedding: for light microscopy, paraffin embedding: Modified Helly’s solution, Bouin’s Modified Helly’s solution, Bouin’s solution, and 10% seawater Formalin solution, and 10% seawater Formalin

for electron microscopy only: for electron microscopy only: Glutaraldehyde/Paraformaldehyde Glutaraldehyde/Paraformaldehyde Solution Solution

Decalcifying & TrimmingDecalcifying & Trimming

Embedding specimens prior to Embedding specimens prior to decalcification using 1~1.5% low-decalcification using 1~1.5% low-melting point agarose or HistoGel melting point agarose or HistoGel (Optional)(Optional)

Decalcifying solutionDecalcifying solutionhydrochloric acid with a chelating hydrochloric acid with a chelating agent, ethylene diamine tetraacetic acid agent, ethylene diamine tetraacetic acid (EDTA) (EDTA)

Processing for Processing for EmbeddingEmbedding

Dehydrate to remove all traces of water Dehydrate to remove all traces of water

Infiltrate with a liquid that can be Infiltrate with a liquid that can be hardened sufficiently to allow cutting of hardened sufficiently to allow cutting of thin section.thin section.

Clear with a reagent that is miscible Clear with a reagent that is miscible with the dehydrating solution and the with the dehydrating solution and the embedding mediumembedding medium

EmbeddingEmbedding

Embedding medium: Embedding medium:

Paraffin for light microscopy &Paraffin for light microscopy &

Glycol methacrylate or epoxy for electron Glycol methacrylate or epoxy for electron microscopymicroscopy

Topology of tissue Topology of tissue (Humason, 1967)

SectioningSectioning Cut sections at 6-10 micrometers or Cut sections at 6-10 micrometers or

thinnerthinner Remove wrinkles and air bubbles from Remove wrinkles and air bubbles from

the ribbon by floating on warm-waterthe ribbon by floating on warm-water A set of sections are picked up on the A set of sections are picked up on the

slide and dried slide and dried

(Humason, 1967)

StainingStaining

To visualize cell and tissue To visualize cell and tissue composition and structurecomposition and structure

Stains are used to distinguish the Stains are used to distinguish the various cell and tissue components; various cell and tissue components; they bind preferentially depending they bind preferentially depending on the biochemistry of the on the biochemistry of the organelles, membranes, etc. organelles, membranes, etc.

StainingStaining ““routine” stain routine” stain

1) Hematoxylin & Eosin1) Hematoxylin & Eosin2) Heidenhain’s Azocarmine-Aniline Blue2) Heidenhain’s Azocarmine-Aniline Blue3) Movat’s Pentachrome3) Movat’s Pentachrome

Special stains are used to identify Special stains are used to identify - particular components or - particular components or microorganismsmicroorganisms- heavy metal impregnation- heavy metal impregnation- immunohistochemistry or - immunohistochemistry or autoradiographyautoradiography

Hematoxylin & Eosin Hematoxylin & Eosin stainingstainingActinodiscus fungiformis

StainingStaining ““routine” stain routine” stain

1) Hematoxylin & Eosin1) Hematoxylin & Eosin2) Heidenhain’s Azocarmine-Aniline Blue2) Heidenhain’s Azocarmine-Aniline Blue3) Movat’s Pentachrome3) Movat’s Pentachrome

Special stains are used to identify Special stains are used to identify - particular components or - particular components or microorganismsmicroorganisms- heavy metal impregnation- heavy metal impregnation- immunohistochemistry or - immunohistochemistry or autoradiographyautoradiography

Heidenhain’s stainingHeidenhain’s stainingGenus Megalactis

(Photo by Dr. Adorian Ardelean)

StainingStaining ““routine” stain routine” stain

1) Hematoxylin & Eosin1) Hematoxylin & Eosin2) Heidenhain’s Azocarmine-Aniline Blue2) Heidenhain’s Azocarmine-Aniline Blue3) Movat’s Pentachrome3) Movat’s Pentachrome

Special stains are used to identify Special stains are used to identify - particular components or - particular components or microorganismsmicroorganisms- heavy metal impregnation- heavy metal impregnation- immunohistochemistry or - immunohistochemistry or autoradiographyautoradiography

Movat’s Pentachrome Movat’s Pentachrome stainingstaining

Astrangia poculata

(photo by Dr. Esther Peters)

StainingStaining ““routine” stain routine” stain

1) Hematoxylin & Eosin1) Hematoxylin & Eosin2) Heidenhain’s Azocarmine-Aniline Blue2) Heidenhain’s Azocarmine-Aniline Blue3) Movat’s Pentachrome3) Movat’s Pentachrome

Special stains are used to identify Special stains are used to identify - particular components or - particular components or microorganismsmicroorganisms- heavy metal impregnation- heavy metal impregnation- immunohistochemistry or - immunohistochemistry or autoradiographyautoradiography

Coral Slide ReadingCoral Slide Reading

The The 2-dimensional2-dimensional images seen in light images seen in light microscopy came microscopy came from from 3-dimensional 3-dimensional structuresstructures

Interpreting the appearance require careful Interpreting the appearance require careful consideration of several factors: what is consideration of several factors: what is known about the specimens, the normal known about the specimens, the normal appearance of the cells and tissues, and appearance of the cells and tissues, and changes that might have affected their changes that might have affected their conditions. conditions.

Coral Slide ReadingCoral Slide Reading

Details and variation of internal Details and variation of internal anatomyanatomy

Reproductive patternReproductive pattern Symbiotic relationshipsSymbiotic relationships BiomechanicsBiomechanics Health of coralsHealth of corals

Get the CHARACTERSto reconstruct the phylogeny!

Actiniaria, Corallimorpharia, Actiniaria, Corallimorpharia, ScleractiniaScleractinia

ActiniariaGenus Megalactis

(Photo by Dr. Adorian Ardelean)

CorallimorphariaCorynactis californica

ScleractiniaAstrangia poculata

(photo by Dr. Esther Peters)

Scleractinian coralsScleractinian corals

ADD MORE PICTURESADD MORE KNOWLEDGEADD MORE CHARACTERS

BETTER CLASSIFICATION & PHYLOGENY

Genus PocilliporaGenus Oculina

Panorama Small Grant, KUNHM & BRCPanorama Small Grant, KUNHM & BRC Graduate Travel Fund, EEB, KUGraduate Travel Fund, EEB, KU NSF grant 9978106 in the program Partnerships to NSF grant 9978106 in the program Partnerships to

Enhance Expertise in Taxonomy (PEET) to Dr. Enhance Expertise in Taxonomy (PEET) to Dr. Daphne Fautin Daphne Fautin

Dr. Esther Peters, Tetra Tech Inc.Dr. Esther Peters, Tetra Tech Inc. Dr. Daphne Fautin, Dr. Meg Daly and Dr. Adorian Dr. Daphne Fautin, Dr. Meg Daly and Dr. Adorian

ArdeleanArdelean

AcknowledgementsAcknowledgements

Vincent B. Hargreaves

Adorian Ardelean George Miller