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Qualification and quantification

of the taste buds of Astyanax


mexicanus

Anti-Calretinin

Teresa Rust
Health Sciences: Physiologic Science
Womens, Gender, and Sexuality Studies
Department of Biological Sciences, University of Cincinnati
Mentor: Dr. Joshua B. Gross
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Outline

Introduction
Research goals
Research methods
Results
Conclusions
Future Work
Acknowledgements
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Introduction

Surface fish

How organisms adapt


to cave environments

Regressive traits
Constructive traits
Cave fish

Surface morphotypes

Cave
Colonization

Cave morphotypes

Common Adaptations:
Behavioral adaptations
Morphological adaptations
Physiological adaptations
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Introduction

Surface fish

Cave fish

https://openi.nlm.nih.gov/detailedresult.php?img=PMC2603666_pgen.1000326.g001&req=4

Anatomy of a taste bud

http://www.accessmedicine.com

Research Goals
1) Qualitative

Visualize taste buds

2) Quantitative

Numerical
Spatial

3) Putative cellular differences

Visualizing taste buds


Calretinin

Electron photomicrograph
of a taste bud without
Calretinin staining
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Visualizing taste buds


Primary Antibody

Primary antibody
attaches to the
taste bud

Visualizing taste buds


Whole Mount

Secondary
antibody
attaches to the
primary antibody

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Visualizing taste buds


Whole Mount

Taste buds
fluoresce red

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Results: Anti-Calretinin staining


Whole Mount

Cave fish

Anti-Calretinin
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Results: Anti-Calretinin staining


Whole Mount

Cave fish
Right

Anti-Calretinin

Surface fish
Right

Anti-Calretinin

Right Axis
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Results: Anti-Calretinin staining


Whole Mount
Surface fish

Cave fish

Dorsal

Dorsal

Cave fish

Anti-Calretinin

Surface fish

Anti-Calretinin

Dorsal Axis
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Results: Anti-Calretinin staining


Whole Mount
Cave fish

Surface fish
Ventral

Ventral

Cave fish

Anti-Calretinin

Surface fish

Anti-Calretinin

Ventral Axis
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Visualizing taste buds


Cross Section

Tissue

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Visualizing taste buds


Cross Section

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Visualizing taste buds


Cross Section

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Visualizing taste buds


Cross Section

Nuclei
fluoresce
blue

Taste buds
fluoresce
red

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Results: Anti-Calretinin staining


Cross Section

Interior of the mouth

Exterior

Anti-Calretinin
Magnification 10x

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Results: Anti-Calretinin staining


Cross Section

Anti-Calretinin
Magnification 20x

Anti-Calretinin
Magnification 40x
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Results: Anti- Calretinin


and Hoechst staining
Cross Section

Anti-Calretinin

Hoechst
Anti-Calretinin

Magnification 63x
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Numerical Analysis
Whole Mount

Cave fish
Right

Anti-Calretinin

Surface fish
Right

Anti-Calretinin

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Numerical Analysis
Whole Mount

Cave fish
Dorsal

Anti-Calretinin

Surface fish
Dorsal

Anti-Calretinin

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Numerical Analysis
Whole Mount

Cave fish
Ventral

Surface fish
Ventral

Anti-Calretinin

Anti-Calretinin

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Results: Cave fish have more


taste buds than surface fish
Number of taste buds

Surface vs cave fish taste buds

Cave fish

Surface fish

1800
1600

1400
1200
1000
800
600
400
200

0
1

22

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Specimen
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Results: Cave fish have more


taste buds than surface fish
Number of taste buds

Average surface vs cave fish taste buds


1400
1400
Mean= 1,100

1200
1200

T-Test:

1000
1000
800800

p = 0.000506

600600

Reject the null hypothesis


that there is no difference
between the number of taste
buds in cave and surface fish

400400

Mean=300.2

200200
00

Cave fish
Surface fish

Surface fish

Cave fish

Specimen
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Spatial Analysis

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Spatial Analysis

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Spatial Analysis

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Spatial Analysis

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Results: Spatial Analysis

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Results: Spatial Analysis


Taste buds are distributed over a greater
area in in cave fish than in surface fish

Cave fish

Surface fish
Left

Left

Left Axis
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Results: Spatial Analysis


Taste buds are distributed over a greater
area in in cave fish than in surface fish

Cave fish
Dorsal

Surface fish
Dorsal

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Results: Spatial Analysis


Taste buds are distributed over a greater
area in in cave fish than in surface fish

Cave fish
Ventral

Surface fish
Ventral

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H3

Cas3

Putative cellular differences

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Visualizing taste buds


Primary Antibody

Primary antibody attaches to the taste bud or


cell undergoing apoptosis or mitosis

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Visualizing taste buds


Secondary Antibody

Secondary antibody
attaches to the primary antibody
Goat Anti-Rabbit
Fluoresces Green

Fluoresces Green

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Visualizing taste buds


Cross Section

Apoptosis
cell death

Mitosis

Taste buds
Nuclei

proliferation

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Results: Putative cellular differences

Hoechst
Anti-Caspase-3
Anti-Calretinin

Apoptosis

Hoechst
Anti-Histone H3
Anti-Calretinin

Mitosis
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Conclusions

Anti-Calretinin

Anti-Calretinin

Cave fish

Anti-Calretinin

Surface fish

Anti-Calretinin

Calretinin
Protein marker for taste
buds in whole mount
and sectioned tissue of
cave fish
Cave fish have more taste
buds than surface fish
Cave fish have taste buds
over a greater area than
surface fish
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Future Work
Genes responsible for the differences
in taste buds
Characteristics of Astyanax mexicanus
caused by the same or different genes

Eye loss
Pigmentation loss
Differences in taste buds
Increased lateral line receptors
Fragmented facial bones

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Acknowledgments
Dr. Joshua Gross
Amanda Powers, Daniel Berning, Andrew
Gangidine, Shane Kaplan, Vivian Varghese,
Stephanie Hacker, Connor Sears, Andrew
Raczka, and Christine Cao
Wise Program, WISE Committee
Funding from:
University of Cincinnati
Department of Biological Sciences
National Science Foundation
Grant DEB-1457630
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WISE
Career goal: Physician
Due to WISE, more interested in
pursuing an MD/Ph.D. program
Next steps:
Research
Volunteer
Shadow
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Questions

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