Qualification and quantification of the taste buds of Astyanax mexicanus

July 29, 2016

Women in Science and Engineering
Research Experiences for Women Undergraduates
Teresa M. Rust
Advisor: Joshua B. Gross, Ph.D.
Department of Biological Sciences
University of Cincinnati

Abstract
Astyanax mexicanus is an ideal species to study aspects of evolution due to the unique
occurrence of parallel evolution. Over time, surface fish have inhabited multiple, separate caves
in Mexico. The surface fish underwent many changes including losing sight, losing
pigmentation, and facial bone fragmentation. In addition, the number and size of taste buds
significantly increased. The intention of this study was to further understand and quantify taste
buds of the surface cave fish through visualizing and analyzing the number, position, and
regeneration taste buds.
Five surface and five cave fish specimens were fixed using 4% PFA and then stained
using the primary antibody 6B3 Anti-Calretinin Antibody (6B3) and secondary antibody Goat
Anti-Mouse Alexa Fluor 546, Texas red. Once imaged using a dissecting microscope and then
analyzed using FIGI software, cave and surface fish specimen 1 and 3 were mounted on slides.
12 m sections were cut and re-stained with 6B3, Goat Anti-Mouse, and fluorescent Hoechst
stain. To explore the putative cellular differences in taste buds, slides were stained with the
primary antibody, Anti-Caspase-3 Antibody (Cas3) or Anti-Histone H3 Antibody (H3), and a
secondary stain of Goat Anti-Rabbit Alexa Fluor 488. The primary antibodies were used to bind
to the different tissue, 6B3 for taste buds, Cas3 for apoptosis, and H3 for mitosis. The secondary
antibodies were used to bind to the primary antibodies to visualize the taste buds, apoptosis, and
mitosis.
Through antibody staining, a 6B3 Anti-Calretinin staining protocol was developed to
better visualize the taste buds and minimize background staining. Cave fish were found to have
significantly more taste buds compared to the surface dwelling morph. Surface fish have taste
buds predominately positioned on their oral epithelium while cave fish have taste buds
2

positioned on their oral epithelium as well as the skin of the maxilla, lower jaw, and ventral
aspect of the head. At present there is not enough information to relate the number of taste buds
undergoing apoptosis and mitosis in cave versus surface fish due to the fact that cave fish have
more taste bud cells than the surface dwelling morph. The comprehensive findings of this
research support the hypothesis that cave fish have more and differently positioned taste buds
than the surface dwelling morph.
Acknowledgments
I would like to sincerely thank and express my appreciation to the people below who
made my research successful and assisted me at each point in the pursuit of my research goals.

This research was supported by my PI, Dr. Gross for his vital support and assistance. His
encouragement and vision made it possible to achieve my research goals. I thank my lab mates
Daniel Berning, Andrew Gangidine, Stephanie Hacker, Shane Kaplan, Amanda Powers, Connor
Sears, Andrew Raczka, and Vivian Varghese who provided insight and training that aided the
research. I would also like to thank Christine Cao for assisting in data collection.

This material is based upon work supported by the National Science Foundation under
Grant No. DEB-1457630.

Table of Contents
Problem Statement
Introduction/Background
Research Methods
Results and Discussion
Conclusion
References
Tables
Figures

Problem Statement
The blind Mexican cave fish, Astyanax mexicanus, lives in the complete darkness of
caves. Although they cannot see, they thrive in an extreme environment. Preliminary research
suggests that cave fish have a greater number of taste buds over a wider area which is thought to
be beneficial when living in complete darkness. In order to be able to better visualize the taste
buds in whole mount and cross sectioned tissue, an improved protocol was designed. The taste
buds were also numerically analyzed through counting the taste buds using FIGI. To more fully
understand how taste buds are positioned on cave fish versus the surface dwelling fish, the taste
buds were spatially analyzed. Another pressing question has been whether taste buds regenerate
more rapidly in cave fish, compared to the surface dwelling fish. So the putative cellular
differences of apoptosis and cellular proliferation were also explored.
Introduction/Background
Astyanax mexicanus, the Mexican tetra, is an ideal organism to use to study how
organisms adapt to cave environments. The Mexican tetra is an organism that has adapted over
time from surface dwelling fish. It is suspected that millions of years ago surface dwelling fish
inhabited caves in Mexico, adapting to the harsh and completely dark environment. (Jeffery
2005, 2009). Organisms, regardless of the landmass that they inhabit, develop very similar
characteristics when colonizing caves. The surface fishs characteristics that have changed over
time to form what is known today as the cave fish are similar to the characteristics of other
organisms that have colonized caves. The adaptations the surface fish underwent over time
formed the cave morph present today in caves throughout Mexico through regressive and
constructive traits. Regressive traits are characteristics that are reduced or absent. Constructive
traits are characteristics that are amplified. Upon first glance, a person looking at Astyanax
5

mexicanus, the Mexican tetra, would immediately note the white or cream color of the Mexican
cave fish. Upon closer examiniation, especially when compared to a surface dwelling fish, one
would notice the lack of eyes. The lack of pigmentation and eyes are both regressive traits. These
striking characteristics of the Mexican cave fish are obvious morphological differences that can
be seen with the naked eye. One would not notice that cave fish have many more taste buds
clustered around their facial structures as compared to the surface fish. This increased number of
taste buds is a constructive characteristic.
It is believed that the absence of eyes and pigment in cave fish is because eye sight and
pigment are extraneous expenses of energy that are unnecessary in complete darkness. (Jeffery
2005, 2009). In addition, cave fish have a larger jaw which is believed to be an advantage to
allow more teeth and taste buds to be present. (Jeffery 2005, 2009; Schemmel, 1980). Both the
Mexican cave fish and surface dwelling morph have neuromasts, sensory organs, to help detect
movement and vibrations in the surrounding environment. Mexican cave fish have an increased
number of neuromasts and it was expected an increased number of taste buds to aid in the search
for food. (Bensouilah and Denizot, 1991; Schemmel, 1975). In their natural cave environment,
cave fish have no known predators and are omnivores. They feed off of debris swept into the
cave and the remains from other cave dwelling organisms. (Culver and Pipan, 2009). In addition
to the increased number of taste buds, different types of taste buds are distributed differently on
cave fish and surface fish, in particular on the lower jaw, with more sensory receptor cells per
taste bud in cave fish. (Boudriot and Reutter, 2001: Varatharasan et al., 2009).
In order to view the taste buds, the tissue must be carefully stained and then placed under
a microscope. The increased number of taste buds, a probable morphological adaptation, is

different from the morphological adaptations mentioned previously, but ultimately shares the
purpose to increase the fitness of the cave fish in its harsh, completely dark environment.
Research Methods
Specimens
The cave and surface fish used in this experiment are native to Mexico. The cave fish were
offspring of specimens from the Pachn cave in Mexico. Specimens were chosen at random and
assigned a number, 1 through 5.
Materials
1% MS-222
Stored at 4C
Fresh 4% Paraformaldehyde (PFA), sterofiltered (100 ml PBS, 4 g PFA, 1 M NaOH and 1 M
HCl to pH balance)
Stored at 4C
Proteinase K
Stored at C
Phosphate Buffered Saline, 10x solution BP-399-20
Fischer Scientific Bioreagents
Triton x-100 for molecular Biology
Sigma Aldrich
10x Phosphate-buffered saline (PBS) (100 ml 100x Phosphate, 900 ml de-ionized water)
Stored at 4C
7

Phosphate-buffered triton (PBT) (1 ml triton, 100 ml 100x Phosphate, 900 ml de-ionized water)
Stored at 4C
Normal Goat Serum
Stored at -18C
6B3 Anti-Calretinin Antibody
Stored at -18C
Cas 3 Anti-Caspase 3 Antibody
Stored at -18C
p-Histone H3 (Ser 10)-R rabbit polyclonal IgG (H3 Anti-Histone 3 Antibody)
Santa Cruz Biotechnology, Stored at 4C
Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L)
Stored at -18C in the dark
Alexa Fluor 546 Goat Anti-Mouse IgG (H+L)
Stored at -18C in the dark
Hoechst
Stored at -4C in the dark
Jung Tissue Freezing Medium
Leica Microsystems
Fluoromount- G

Sothern Biotech
Procedure
Throughout the whole process, gloves were worn.
Full Body Specimen: Abstaining
Ten specimens were randomly collected from the tank, 5 surface fish and 5 cavefish,
recording the tank number. About 100 ml of 1% MS-222 was poured into the 1 liter tank
containing the specimens. Specimens were undisturbed for 15-20 minutes, until the gills of the
specimens stopped moving. Each specimen was placed in a glass vial and covered with 4%
Paraformaldehyde solution (PFA). The vials were placed on the nutator mixer for 2 hours at
room temperature. The specimens were then rinsed three times using 10x Phosphate-buffered
saline (PBS). The rinse solution was extracted with a transfer pipette and then enough PBS was
added to cover the specimen. The vials were placed on the nutator mixer for five minutes. A
protein digest was then performed. In each vial, the PBS rinse solution was extracted and the
digest solution was added. 1000 l of PBS and 10 l of proteinase K, per specimen solution was
prepared. The proteinase K was drawn into the pipette very slowly and directly expelled into the
PBS due to its viscosity. Each specimen was just covered with the digest solution. The vials were
then placed on the nutator mixer for 15 minutes due to the small size of the specimens. The
specimens were then rinsed three times. The rinse solution was extracted with a transfer pipette
and then enough PBS was added to cover the specimen. The vials were placed on the nutator
mixer for five minutes. The specimen was re-fixed using the same amount of 4% PFA as was
used previously for 15 minutes. The specimens were rinsed three times. The rinse solution was
extracted with a transfer pipette and then enough PBS was added to cover the specimen. The

vials were placed on the nutator mixer for ten minutes. The specimens were then rinsed three
times. The rinse solution was extracted with a transfer pipette and then enough Phosphatebuffered triton (PBT) was added to cover the specimen. The vials were placed on the nutator
mixer for ten minutes. An overnight primary antibody cocktail was made (1:2000 dilution) 6B3
Anti-Calretinin antibody in PBT with 5% total volume Normal Goat Serum. For 10 small
specimens the cocktail consisted of 12000 l PBT, 6 l 6B3, and 600 l Normal Goat Serum.
Each specimen was covered with just enough solution to be submerged. The vials were placed on
the nutator mixer for two nights at room temperature. The specimens were then rinsed five times.
The rinse solution was extracted with a transfer pipette and then enough PBT was added to cover
the specimen. The vials were placed on the nutator mixer for five minutes. The specimens were
rinsed five times. The rinse solution was extracted with a transfer pipette and then enough PBT
was added to cover the specimen. The vials were placed on the nutator mixer for ten minutes. An
overnight secondary antibody stain was made (1:300 dilution) Goat Anti-Mouse alexa fluor 546
in PBT with 5% total volume Normal Goat Serum in the dark. For 10 small specimens the
cocktail consisted of 10,000 l PBT, 6 l 33.4 l Goat Anti-Mouse alexa flour 546, and 500 l
Normal Goat Serum. Each specimen was covered with just enough solution to be submerged.
The vials were wrapped in aluminum foil and placed on the nutator mixer for one night at room
temperature. The specimens were then rinsed five times. The rinse solution was extracted with a
transfer pipette and then enough PBT was added to cover the specimen. The vials were placed on
the nutator mixer for five minutes. The specimens were rinsed five times. The rinse solution was
extracted with a transfer pipette and then enough PBT was added to cover the specimen. The
vials were placed on the nutator mixer for ten to sixty minutes. The rinse PBT solution was

10

drawn off, and PBS was added to the vial to cover the specimen. The specimens were then
imaged. The vials were placed at 4C when not being imaged.
Imaging
The specimens were imaged using a Leica M205 FA dissecting microscope with C mount
0.70x, Leica DFC310 FX, and fluorescence attachments. The dorsal and ventral axes were
imaged at 30x magnification, positioned anterior left and posterior right. The lateral left and right
axes were imaged at 25x magnification. The specimens were held into place while imaging using
a cut agar block in a plastic petri dish. Montages were taken of each axis using Texas red
Fluorescence.
Cross Section: Abstaining
Cryosectioning
The preserved specimen was obtained from the refrigerator. The head of each specimen
was cut in a plastic petri dish under a dissecting microscope using a scalpel. The head of the
specimen was mounted in a cryostat model with tissue freezing medium. The lips of the
specimen were positioned with forceps at the bottom of the cryostat model. The cryostat model
was placed with mounted specimen in the Leica CM 1860 cryostat to freeze. The temperature
was set at 18C and the thickness of the sections was set to 12m. Once the tissue freezing
medium was frozen and opaque in color, a razor blade was used to make a notch in the plastic.
The frozen block of tissue freezing medium was ejected out of the cryostat model. The opaque
block of frozen tissue freezing medium was placed onto a chuck. The tissue freezing medium
block containing the tissue was attached to the chuck by adding tissue freezing medium in a
circular motion around the circumference of the opaque block of frozen tissue freezing medium.
11

The clear tissue freezing medium froze, turning opaque and lost its shine, signifying that it was
frozen. The chuck was placed in the specimen holder. Use the arrow buttons on the left side of
the instrument panel were used to bring the specimen closer to the blade. The angle of the
specimen was oriented to the razor blade so that smooth thin layers of tissue were sectioned. The
wheel positioned on the right of the cryostat machine was used to bring the frozen tissue
forward, resulting in 12 m sections of tissue. Clean glass slides were positioned over top of the
tissue resulting in the tissue adhering to the slide. For cave fish, 22-26 slides were used per
specimen. For surface fish 18-20 slides were used per specimen. The slides were allowed to dry
for 48 hours then abstained.
Abstaining
Each slide was blocked in a solution of Phosphate-buffered triton (PBT) and 5% Normal
Goat Serum. For 10 slides, 3,000 l of PBT was used and 150 l of Normal Goat Serum. An
automatic pipette was used to distribute 250 l of the solution onto each slide. A piece of
parafilm, cut to the size of the slide, was laid on the slide with forceps and each slide was placed
in a plastic air tight container. The plastic air tight container was prepared with a layer of damp
paper towels underneath well plates that contained about a centimeter of water. The slides were
blocked, undisturbed, for two hours at room temperature.
The parafilm was carefully removed with forceps. The slides were dried by blotting the
edge and wiping the back of the slide on a paper towel. Then a dilution (5:1000) of primary
antibody stain of 6B3 Anti-Calretinin, Anti-Caspase (Cas3) or Anti-Histone (H3), PBT, and 5%
Normal Goat Serum was prepared. For 10 slides, 3,000 l of PBT, 15 l of 6B3, 15 l of Cas3 or
15 l H3 and 150 l of Normal Goat Serum was used. An automatic pipette was used to
distribute 250 l of the solution onto each slide. For the control, no primary antibody was
12

introduced to the tissue on the slide. A piece of parafilm, cut to the size of the slide, was laid on
top the slide with forceps and each slide was placed in a plastic air tight container. The plastic air
tight container was prepared with a layer of damp paper towels underneath well plates that
contained about a centimeter of water. The slides were left undisturbed overnight at 4C.
The parafilm was carefully removed with forceps. The slides were dried by blotting the
edge and wiping the back of the slide on a paper towel. A vertical glass staining dishes were used
to rinse and wash the slides. The rinse was performed by pouring in an equal amount (50:50) of
PBS and PBT into the vertical glass staining dishes so that all of the tissue on the slides was
covered with the solution. The slides were placed in the vertical glass staining dishes without
touching and then removed. Six washes were then performed. The slides were dried by blotting
the edge and wiping the back of the slide on a paper towel. The vertical glass staining dishes
were rinsed with deionized water and an equal amount (50:50) of PBS and PBT, the same
amount as before, was poured into the vertical glass staining dishes. The slides were reinserted
and placed on a shake table for 30 minutes.
After the sixth wash was performed, the slides were removed from the vertical glass
staining dishes and then dried by blotting the edge and wiping the back of the slide on a paper
towel. The slides were stained with a dilution of 1:1000, Goat Anti-Mouse 546 and PBT. For ten
slides, 3,000 l of PBT and 3 l of Goat Anti-Mouse 546 were used. An automatic pipette was
used to distribute 250 l of the solution onto each slide. A piece of parafilm, cut to the size of the
slide, was laid onto the slide with forceps and each slide was placed in a plastic air tight
container. The plastic air tight container was prepared with a layer of damp paper towels
underneath well plates that contained about a centimeter of water. The slides were left
undisturbed overnight at 4C.
13

The parafilm was carefully removed with forceps. The slides were dried by blotting the
edge and wiping the back of the slide on a paper towel. A vertical glass staining dishes were used
to rinse and wash the slides. The rinse was performed by pouring in an equal amount (50:50) of
PBS and PBT into the vertical glass staining dishes so that all of the tissue on the slides was
covered with the solution. The slides were placed in the vertical glass staining dishes without
touching and then removed. Six washes were then performed. The slides were dried by blotting
the edge and wiping the back of the slide on a paper towel. The vertical glass staining dishes
were rinsed with deionized water and an equal amount (50:50) of PBS and PBT, the same
amount as before, was poured into the vertical glass staining dishes. The slides were reinserted
and placed on a shake table for 30 minutes.
After the sixth wash was performed, the slides were removed from the vertical glass
staining dishes and then dried by blotting the edge and wiping the back of the slide on a paper
towel. The slides were stained with a dilution of 1:1000, Goat Anti-Rabbit 488 and PBT. For ten
slides, 3,000 l of PBT and 3 l of Goat Anti-Rabbit 488 were used. An automatic pipette was
used to distribute 250 l of the solution onto each slide. A piece of parafilm, cut to the size of the
slide, was laid onto the slide with forceps and each slide was placed in a plastic air tight
container. The plastic air tight container was prepared with a layer of damp paper towels
underneath well plates that contained about a centimeter of water. The slides were left
undisturbed overnight at 4C.
The parafilm was carefully removed with forceps. The slides were dried by blotting the
edge and wiping the back of the slide on a paper towel. A vertical glass staining dishes were used
to rinse and wash the slides. The rinse was performed by pouring in an equal amount (50:50) of
PBS and PBT into the vertical glass staining dishes so that all of the tissue on the slides was
14

covered with the solution. The slides were placed in the vertical glass staining dishes without
touching and then removed. Six washes were then performed. The slides were dried by blotting
the edge and wiping the back of the slide on a paper towel. The vertical glass staining dishes
were rinsed with deionized water and an equal amount (50:50) of PBS and PBT, the same
amount as before, was poured into the vertical glass staining dishes. The slides were reinserted
and placed on a shake table for 30 minutes.
After the sixth wash was performed, the slides were removed from the vertical glass
staining dishes and then dried by blotting the edge and wiping the back of the slide on a paper
towel. The slides were stained with a dilution of 1:1000, Hoechst and PBT. For ten slides, 3,000
l of PBT and 3 l of Hoechst were used. An automatic pipette was used to distribute 250 l of
the solution onto each slide. A piece of parafilm, cut to the size of the slide, was laid onto the
slide with forceps and each slide was placed in a plastic air tight container. The plastic air tight
container was prepared with a layer of damp paper towels underneath well plates that contained
about a centimeter of water. The slides were left undisturbed for 30 minutes at room temperature.
The parafilm was carefully removed with forceps. The slides were dried by blotting the
edge and wiping the back of the slide on a paper towel. Five drops of fluoromount were placed
on each slide using a transfer pipette. The cover slides were then positioned on the slides using
forceps. The slides were allowed to dry overnight and then imaged using a photomicroscope.

Imaging

15

The specimens were imaged using a Leica DM 2000 LED microscope with C mount
0.70x, Leica DFC310 FX, Leica DMC 4500, and fluorescence attachments. The tissue was
imaged at 10x, 20x, 40x, and 63x magnification using Texas Red Fluorescence (TXR), Green
Fluorescent Protein (GFP), and 4, 6-diamidino-2-phenylindole (DAPI).
Numerical Analysis
The numerical analysis was completed on a Macintosh desktop computer using FIGI. The
photomicrograph images of the axes were dragged individually into FIGI. The Multi-point tool
was selected. The settings were then selected. Type (crosshair), color (yellow), size
(small), and label points was unchecked. A crosshair was placed on each taste bud by using the
mouse to click once. The counter shows the number of taste buds counted. The number was
recorded in excel in the proper category, related to the specimen and axis, to be later analyzed.
The taste buds were categorized as calretinin ++ and calretinin +. Calretinin ++ structures
were determined to be taste buds due to their very bright calretinin positive, red fluorescence.
Calretinin + structures were determined to be putative taste buds due to their dimmer, but still
bright calretinin positive, red fluorescence.
Spatial Analysis
The spatial analysis was completed on a Macintosh desktop computer using power point
and Adobe Photoshop CS6. The photomicrograph images of the axes were dragged individually
into power point. The proportions of the image were kept constant, resizing the image so that it
filled the entire slide. "Shapes," "lines and connectors," and "curve" were selected in power point
to draw an outline over each specimen. Format, picture, picture, brightness, and
contrast was used to change the contrast and brightness of the photomicrograph. The contrast
and brightness were increased with each image and then returned to the original coloring once
16

the outline was drawn. The line was selected and "format" "shape" and then "line" was selected.
The color was changed to black, weight to 2pt, and style to a solid line. The photomicrograph
image was deleted. The outline was saved as a pdf.
Adobe photoshop CS6 was used to create two layers and labeled accordingly. The
outline pdf was dragged into photoshop. Each layer was selected using the layers tab and the
opacity was decreased until both layers could be seen. The "Move tool v" (on the left hand side
of the screen the top selection of an arrow and crosshair) was selected, allowing the image to be
moved. The outline and image were aligned. In order to increase the ability to see the taste buds,
"adjustments" then "brightness/contrast" was increased. The amount of increase depended on the
exposure of the image. Two new layers were added, "calretinin +" and "calretinin ++" the
calretinin ++ layer was selected. The tools to mark each taste bud were selected. "Pencil tool b"
was selected from the left hand side. "Brush" (on the right hand side of the screen) size was
selected. "Hard round 30," "20 px," roundness 100%," hardness 100%," and spacing 25%"
were selected. The color of the brush was changed using color picker (on the left hand size).
The color of calretinin ++ was ff0000, a red color. The color of calretinin + was 020e68, a dark
blue color.
To obtain overlays of all of the axes, screen shots of the outlines with taste bud markers
were taken using command shift 4 and were dragged into Photoshop, different layers
labeled accordingly and the opacity was decrease on each layer until all the layers were able to
be seen.

Results and Discussion

17

To visualize taste buds in cave fish and surface dwelling fish, fluorescent
immunohistochemistry was used in cross sectioned tissue as well as whole specimens. Cross
sectioned tissue was examined to view the structure of the taste buds inside and outside of the
mouth of each specimen. Whole mounted tissue was used to view taste buds and their
distribution on the outside of the mouth and face of the specimens.
Using 6B3 Anti-Calretinin (at a concentration of 0.5%) and Goat Anti-Mouse 546 (at a
concentration of 0.1%), taste buds fluoresced red when exposed to ultraviolet light. The Hoechst
stain (at a concentration of 0.1%) was successfully used to see the nuclei in the tissue, most
relevant to this research in the taste bud cells. The onion or head of garlic shape of the taste buds
was viewed in the cross sectioned tissue. At 10 times magnification, see fig. 8 and fig. 9, the
taste buds could be viewed inside of the mouth and on the outside of the lips and face of the cave
fish. Some taste buds were seen on the outside of the lips and inside of the mouth of the surface
dwelling fish. At 20 times magnification, see fig. 10, the clusters of taste buds can be seen. The
onion or head of garlic shape of taste buds becomes easier to distinguish, see fig. 10. At 40 times
magnification, the onion or head of garlic shape of taste buds is seen. The individual cells in the
taste buds begin to come into focus, see fig. 11. At 63 times magnification, the onion or head of
garlic shape of the taste bud is very distinct along with the individual cells that form the taste
bud. Image overlay was used to take an overlapping photomicrograph of the taste bud (TXR) and
the Hoechst stain (DAP). Fig. 12 shows the nuclei in the taste bud, reinforcing the conclusion
that 6B3 Anti-Calretinin (at a concentration of 0.5%) and Goat Anti-Mouse 546 (at a
concentration of 0.1%) is a valuable protein stain for taste bud cells. Taste bud cells contain
nuclei and taste buds have a head of garlic or onion shape. The presence of stained nuclei in the
onion shaped taste bud, supports the hypothesis that taste buds were stained using 6B3 Anti-

18

Calretinin (at a concentration of 0.5%) and Goat Anti-Mouse 546 (at a concentration of 0.1%).
See fig. 27 and 28, there was a greater number of cells in the cave fish as opposed to the surface
fish that fluoresced green when exposed to ultraviolet light. This indicates that more cells
undergo apoptosis and mitosis in cave fish than surface fish, but it is not known at this time if it
is due to the greater number of taste buds present in cave fish.
To numerically analyze and visualize taste buds in cave fish and surface dwelling fish
whole specimens were stained using the primary antibodies 6B3 Anti-Calretinin (at a
concentration of 0.05%) as well as the secondary antibody Goat Anti-Mouse 546, taste buds
fluoresced red when exposed to ultraviolet light. The dorsal, ventral, anterior, and posterior axis
images taken of each specimen, see fig. 2 through 7. The total number of taste buds per specimen
was recorded, see fig. 14 through 20. The average total number of taste buds on surface fish is
300.2 and cave fish is 1100, see fig. 21. A one tailed two-sample t-Test assuming unequal
variances was performed. The null hypothesis stating that there is no difference between the
number of taste buds in cave and surface fish was rejected with a p value of p = 0.000506. Cave
fish have significantly more taste buds than surface fish. The images of the whole mounted
specimens were used and outlines were created to visualize the distribution of the taste buds, see
fig. 22 through 27. These outlines depict the clusters of taste buds so the surface fish and cave
fish taste bud distribution can be compared. Cave fish have taste buds located in dense clusters
on their oral epithelium, maxilla, lower jaw, and ventral aspect of the head while the surface
dwelling morph has taste buds clusters concentrated on the oral epithelium.

19

Conclusion
The taste buds of Astyanax mexicanus, the Mexican cave fish, and the surface dwelling
morph were visualized and analyzed. Through numerical and spatial analysis, the hypothesis that
cave fish have more taste buds dispersed throughout the facial structure was supported. The
average number of taste buds on the exterior of a surface fish is 300.2 while for cave fish it is
1,100 taste buds. Cave fish have taste buds located on their oral epithelium as well as the
maxilla, lower jaw, and ventral aspect of the head while the surface dwelling morph has taste
buds concentrated along the oral epithelium. Cave fish have more cells in taste buds undergoing
apoptosis and mitosis, but at present there is not enough information to confidently make the
conclusion that cave fish have more cell bereavement and restoration. This conclusion cannot yet
be made due to the greater number of taste buds that cave fish have as opposed to the surface
dwelling morph. The congregate findings support the overall hypothesis that cave fish have more
taste buds found on more positions of the facial structure than does the surface dwelling morph.

References
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Bensouilah, M., Denizot, J.P., 1991. Taste buds and neuromasts of Astyanax jordani: distribution
and immunochemical demonstration of co-localized substance P and enkephalins. Eur. J.
Neu-rosci. 3, 407414.

Boudriot, F., Reutter, K., 2001. Ultrastructure of the taste buds in the blind cave fish Astyanax
jor-dani (Anoptichthys) and the sighted river fish Astyanax mexicanus (Teleostei,
Characidae). J. Comp. Neurol. 434, 428444.

Culver, D.C., Pipan, T., 2009. The Biology of Caves and Other Subterranean Habitats. Oxford
University Press, Oxford.

Jeffery, W.R., 2005. Adaptive evolution of eye degeneration in the Mexican blind cavefish. J.
Hered. 96, 185196.

Jeffery, W.R., 2009a. Evolution and development in the cavefish Astyanax. Curr. Top. Dev.
Biol. 86, 191221.

Jeffery, W.R., 2009b. Regressive evolution in Astyanax cavefish. Annu. Rev. Genet. 43, 2547.

Schemmel, C., 1980. Studies on the genetics of feeding behaviour in the cave fish Astyanax
mexicanus f. Anoptichthys. Z. Tierpsychol. 53, 922.
Varatharasan, N., et al., 2009. Taste bud development and patterning in sighted and blind morphs
of Astyanax mexicanus. Dev. Dyn. 238, 30563064.

Figures

21

Figure 1. Surface dwelling morph and Mexican cave fish

Anti-Calretinin
Figure 2. Cave fish right axis. 25x magnification

Anti-Calretinin

22

Figure 3. Surface dwelling morph right axis. 25x magnification

Anti-Calretinin
Figure 4. Cave fish dorsal axis. 30x magnification

Anti-Calretinin
Figure 5. Surface dwelling morph dorsal axis. 30x magnification.

23

Anti-Calretinin
Figure 6. Cave fish ventral axis. 30x magnification.

Anti-Calretinin
Figure 7. Surface dwelling morph ventral axis. 30x magnification

24

Figure 8. Cave fish taste buds. 10x magnification

Figure 9. Cave fish taste buds. 10x magnification

Anti-Calretinin
Figure 10. Cave fish taste buds. 20x magnification

25

Anti-Calretinin
Figure 11. Cave fish taste buds. 40x magnification

Figure 12. Cave fish taste buds. 63x magnification

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Figure 13. Cave fish taste buds and nuclei. 63x magnification

Figure 14. Cave fish right axis. 25x magnification

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Figure 15. Surface-dwelling morph right axis. 25x magnification

Figure 16. Cave fish dorsal axis. 30x magnification

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Figure 17. Surface-dwelling morph dorsal axis. 30x magnification

Figure 18. Cave fish ventral axis. 30x magnification

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Figure 19. Surface-dwelling morph dorsal axis. 30x magnification

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Figure 20. Specimens, total number of taste buds.

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Figure 21. Specimens, total number of taste buds.

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Figure 22. Image overlay cave fish left axis

Figure 23. Image overlay surface-dwelling morph left axis

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Figure 24. Image overlay cave fish dorsal axis

Figure 25. Image overlay surface-dwelling morph dorsal axis

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Figure 26. Image overlay cave fish ventral axis

Figure 27. Image overlay surface-dwelling morph ventral axis

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Figure 27. Cave fish nuclei, mitosis, and taste buds. 40x magnification

Figure 28. Cave fish nuclei, apoptosis, and taste buds. 40x magnification

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