Understanding Coral Histology - How & Why - Ha-Rim Cha Division of Invertebrate Zoology The 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 Corallimorpharia Cylindrical body Family Sideractiidae Family Corallimorphidae Plate-like body Family Ricordeidae Family Discosomatidae  Questions about relationships within the order Vincent B. Hargreaves

 Questions about relationships between Corallimorpharia and two closest orders Actiniaria (sea anemones) and Scleractinia (stony corals) ↑ Stony coralCorallimorpharia ↑↑ Sea anemones Phylogeny of Corallimorpharia

Characters to be examined 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 Coral 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 Coral Taxonomy & Phylogeny Taxonomy of scleractinian corals is based on their skeletal structure Taxonomy of scleractinian corals is based on their skeletal structure Brin Edwards

Taxonomy of scleractinian corals Taxonomy of scleractinian corals Genus Fungia Genus Fungia Genus Tubastrea Genus Tubastrea Characters used for Coral Taxonomy & Phylogeny

Order level phylogeny of Hexacorallia Order level phylogeny of Hexacorallia ? Scleractinia Actiniaria Corallimorpharia

Phylogeny Order level phylogeny of Hexacorallia Order level phylogeny of Hexacorallia a. 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 Anatomy Additional characters on coral morphology Additional characters on coral morphology An independent character set to compare with published molecular data An independent character set to compare with published molecular data

Histology Histology (= Microscopic anatomy) is… Histology (= Microscopic anatomy) is… the study of cells, tissues, organs, and organ systems of plants and animals the study of cells, tissues, organs, and organ systems of plants and animals

Histology Histology (= Microscopic anatomy) is… Histology (= Microscopic anatomy) is… the study of cells, tissues, organs, and organ systems of plants and animals the study of cells, tissues, organs, and organ systems of plants and animals Structure & Composition of an organism or Structure & Composition of an organism or a part reflects its key functions a part reflects its key functions (e.g. respiration, metabolism, reproduction, movement) (e.g. respiration, metabolism, reproduction, movement)

Coral Histology To support studies on ecology, physiology, reproduction, biochemistry, immunology, embryology, and systematics To support studies on ecology, physiology, reproduction, biochemistry, immunology, embryology, and systematics Histopathology -- Study on coral diseases (e.g. white-band disease, black-band disease, red- band disease) or bleaching event Histopathology -- Study on coral diseases (e.g. white-band disease, black-band disease, red- band disease) or bleaching event

Bleaching & Coral Disease Black-Band Disease ( photos by E. Peters) Yellow-Band Disease Bleaching ALIVE! DEAD

Procedure for coral histology Collecting Fixation Decalcifying and Trimming Embedding Sectioning Staining

Specimen Collection Using a cold chisel and a coring device (for a large colony) Using a cold chisel and a coring device (for a large colony) Relax the collected specimens Relax the collected specimens Obtaining permission prior to conducting the study Obtaining permission prior to conducting the study Reporting export and import of collected materials to the United States Fish and Wildlife Service is required Reporting export and import of collected materials to the United States Fish and Wildlife Service is required

Fixation To preserve the structure and composition of the tissue To preserve the structure and composition of the tissue Post-mortem effects are usually prevented by denaturing and precipitating protein, or by cross-linking the proteins with aldehyde and/or other chemicals Post-mortem effects are usually prevented by denaturing and precipitating protein, or by cross-linking the proteins with aldehyde and/or other chemicals

Fixation The choice of fixative will depend on the embedding medium to be used. The choice of fixative will depend on the embedding medium to be used. for light microscopy, paraffin embedding: Modified Helly’s solution, Bouin’s solution, and 10% seawater Formalin for light microscopy, paraffin embedding: Modified Helly’s solution, Bouin’s solution, and 10% seawater Formalin for electron microscopy only: Glutaraldehyde/Paraformaldehyde Solution for electron microscopy only: Glutaraldehyde/Paraformaldehyde Solution

Decalcifying & Trimming Embedding specimens prior to decalcification using 1~1.5% low-melting point agarose or HistoGel (Optional) Embedding specimens prior to decalcification using 1~1.5% low-melting point agarose or HistoGel (Optional) Decalcifying solution hydrochloric acid with a chelating agent, ethylene diamine tetraacetic acid (EDTA) Decalcifying solution hydrochloric acid with a chelating agent, ethylene diamine tetraacetic acid (EDTA)

Processing for Embedding Dehydrate to remove all traces of water Dehydrate to remove all traces of water Infiltrate with a liquid that can be hardened sufficiently to allow cutting of thin section. Infiltrate with a liquid that can be hardened sufficiently to allow cutting of thin section. Clear with a reagent that is miscible with the dehydrating solution and the embedding medium Clear with a reagent that is miscible with the dehydrating solution and the embedding medium

Embedding Embedding medium: Embedding medium: Paraffin for light microscopy & Glycol methacrylate or epoxy for electron microscopy Topology of tissue Topology of tissue (Humason, 1967)

Sectioning Cut sections at 6-10 micrometers or thinner Cut sections at 6-10 micrometers or thinner Remove wrinkles and air bubbles from the ribbon by floating on warm-water Remove wrinkles and air bubbles from the ribbon by floating on warm-water A set of sections are picked up on the slide and dried A set of sections are picked up on the slide and dried (Humason, 1967)

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

Staining “routine” stain 1) Hematoxylin & Eosin 2) Heidenhain’s Azocarmine-Aniline Blue 3) Movat’s Pentachrome “routine” stain 1) Hematoxylin & Eosin 2) Heidenhain’s Azocarmine-Aniline Blue 3) Movat’s Pentachrome Special stains are used to identify - particular components or microorganisms - heavy metal impregnation - immunohistochemistry or autoradiography Special stains are used to identify - particular components or microorganisms - heavy metal impregnation - immunohistochemistry or autoradiography

Hematoxylin & Eosin staining Actinodiscus fungiformis

Staining “routine” stain 1) Hematoxylin & Eosin 2) Heidenhain’s Azocarmine-Aniline Blue 3) Movat’s Pentachrome “routine” stain 1) Hematoxylin & Eosin 2) Heidenhain’s Azocarmine-Aniline Blue 3) Movat’s Pentachrome Special stains are used to identify - particular components or microorganisms - heavy metal impregnation - immunohistochemistry or autoradiography Special stains are used to identify - particular components or microorganisms - heavy metal impregnation - immunohistochemistry or autoradiography

Heidenhain’s staining Genus Megalactis (Photo by Dr. Adorian Ardelean)

Staining “routine” stain 1) Hematoxylin & Eosin 2) Heidenhain’s Azocarmine-Aniline Blue 3) Movat’s Pentachrome “routine” stain 1) Hematoxylin & Eosin 2) Heidenhain’s Azocarmine-Aniline Blue 3) Movat’s Pentachrome Special stains are used to identify - particular components or microorganisms - heavy metal impregnation - immunohistochemistry or autoradiography Special stains are used to identify - particular components or microorganisms - heavy metal impregnation - immunohistochemistry or autoradiography

Movat’s Pentachrome staining Astrangia poculata (photo by Dr. Esther Peters)

Staining “routine” stain 1) Hematoxylin & Eosin 2) Heidenhain’s Azocarmine-Aniline Blue 3) Movat’s Pentachrome “routine” stain 1) Hematoxylin & Eosin 2) Heidenhain’s Azocarmine-Aniline Blue 3) Movat’s Pentachrome Special stains are used to identify - particular components or microorganisms - heavy metal impregnation - immunohistochemistry or autoradiography Special stains are used to identify - particular components or microorganisms - heavy metal impregnation - immunohistochemistry or autoradiography

Coral Slide Reading The 2-dimensional images seen in light microscopy came from 3-dimensional structures The 2-dimensional images seen in light microscopy came from 3-dimensional structures Interpreting the appearance require careful consideration of several factors: what is known about the specimens, the normal appearance of the cells and tissues, and changes that might have affected their conditions. Interpreting the appearance require careful consideration of several factors: what is known about the specimens, the normal appearance of the cells and tissues, and changes that might have affected their conditions.

Coral Slide Reading Details and variation of internal anatomy Details and variation of internal anatomy Reproductive pattern Reproductive pattern Symbiotic relationships Symbiotic relationships Biomechanics Biomechanics Health of corals Health of corals Get the CHARACTERS to reconstruct the phylogeny!

Actiniaria, Corallimorpharia, Scleractinia Actiniaria Genus Megalactis (Photo by Dr. Adorian Ardelean) Corallimorpharia Corynactis californica Scleractinia Astrangia poculata (photo by Dr. Esther Peters)

Scleractinian corals ADD MORE PICTURES ADD MORE KNOWLEDGE ADD MORE CHARACTERS BETTER CLASSIFICATION & PHYLOGENY Genus PocilliporaGenus Oculina

Panorama Small Grant, KUNHM & BRC Panorama Small Grant, KUNHM & BRC Graduate Travel Fund, EEB, KU Graduate Travel Fund, EEB, KU NSF grant in the program Partnerships to Enhance Expertise in Taxonomy (PEET) to Dr. Daphne Fautin NSF grant in the program Partnerships to Enhance Expertise in Taxonomy (PEET) to Dr. Daphne Fautin Dr. Esther Peters, Tetra Tech Inc. Dr. Esther Peters, Tetra Tech Inc. Dr. Daphne Fautin, Dr. Meg Daly and Dr. Adorian Ardelean Dr. Daphne Fautin, Dr. Meg Daly and Dr. Adorian Ardelean Acknowledgements Vincent B. Hargreaves Adorian ArdeleanGeorge Miller