Comparison of Cutaneous Respiration Rates of Mudskippers (Periophthalmus) and
Amphibians (Xenopus, Bombina, and Pseudacris) of Varied Ecology
Chantal Eidelstein, Zachary Perry, and Azin Saebi
Department of Biological Sciences
Saddleback College
Mission Viejo, California 92692
Cutaneous respiration in selected vertebrates allow for animals to conduct
metabolism while submerged under water. These animals tend to flourish in wet
environments that provide their skin with proper moisture. It would be logical to
hypothesize that an animal with a more aquatic lifestyle would exhibit higher
cutaneous respiration rate as well as overall respiration rate. The rate at which an
amphibian respires cutaneously and the proportion between its cutaneous and
active respiration rates is largely dependent the species’ ecology. Mudskippers have
evolved cutaneous respiratory patterns similar to those of amphibians that exhibit
comparable semiaquatic lifestyles. In this experiment, the weights of each of the five
Fire-bellied Toads (Bombina orientalis), five African Dwarf Frogs (Hymenochirus
boettgeri), five Pacific Tree Frogs (Pseudacris regilla), and two mudskipper species
(Periophthalmus barbarous and novemradiatus) were recorded before submerging
them into either a capped jar filled with water or a dry jar attached to a PASCO
CO2 probe. At room temperature, the animals metabolized at specified times
thought to conclude the most accurate results. It was discovered that the African
Dwarf frogs (Hymenochirus boettgeri) had the highest cutaneous respiration rate.
The sample selected for terrestrial data, Pacific Tree frogs (Pseudacris regilla), had
the lowest ratio and therefore became the terrestrial model. The animals gave
sufficient evidence to show a correlation between evolutionary pathways of
cutaneous respiration.
Introduction
All vertebrates conduct at least
some of the respiration cutaneously, but
amphibians rely one this method more
than most; it may account for up to
100% of total aerial respiration in some
groups (plethodontid salamanders) and it
is the sole viable mode of respiration for
most adult amphibians when they are
submerged (Piiper 1982, Joseph 1998).
Terrestrial
species
from
desert
environments will have evolved
mechanisms that limit their cutaneous
respiration in order to prevent
evaporative water loss, while aquatic
species from the tropics can be expected
to have relatively high cutaneous
respiration rates so that they can remain
active when submerged for long periods
(Piiper 1982).
In an interesting evolutionary
twist, one group of ray-finned fish has
evolved a similar semiaquatic lifestyle to
many amphibians and, like them, relies
heavily on cutaneous respiration. These
are
the
mudskippers
(gobiidae:
oxudercinae), which can be found along
the coasts of Africa, southern Asia,
Indonesia, and northern Australia where
the inhabit mangroves swamps and
coastal mudflats (Aguilar 2000, Alderton
2008, Gordon 1978, Teal 1967). These
highly specialized fish spend much of
their time on land, provided they can
keep their skin moist, where they respire
through their skin and the lining of their
mouths and throats; they will drown if
they remain underwater too long, so they
use their gills, not to actively respire like
other fish, but to simply hold a bubble of
air (Aguilar 2000, Teal 1967). This is in
contrast to other air-breathing fish, such
as
lungfish,
polypterids,
and
anabantoids, which either gulp air at the
surface through their mouths or via
spiracles (Jeffrey 2014, Piiper 1982).
While amphibians are descended from
fish, mudskippers are advanced rayfinned fish (perciforme actinopterygians)
and therefore are far removed from the
group
that
contains
amphibians
(sarcopterygians) by well over 350
million years of evolution (Jeffrey
2014).
With so much convergent
evolution at play, it would be expected
that the cutaneous respiration rate of
mudskippers would approach that
exhibited by amphibians that live under
similar conditions, i.e.: those species that
are semiaquatic, provided they are at the
same temperature (both are ectotherms).
To test this hypothesis, a selection of
amphibians including the terrestrial
Pseudacris regilla (Stebbins 2003), the
semiaquatic Bombina orientalis (Bartlett
2010), and the aquatic Hymenochirus
boettgeri (Bartlett 2010), will be subject
to a series of experiments and their
results will be compared to that of a
mudskipper (Periophthalmus sp.) to
determine if the mudskippers’ cutaneous
respiration qualities have convergently
approached those of the amphibian with
the most similar ecology: Bombina. The
tests include submerging the specimens
in water for a specified length of time
and then testing the water’s dissolved
oxygen content using a Winkler titration
to determine their aquatic cutaneous
respiration rate, and measuring the
specimens’ aerial CO2 production rate
via a probe in order to determine their
overall respiration rate. These results
will be compared and contrasted with
each other to confirm or deny the
hypothesis.
Materials and Methods
Five
Fire-bellied
Toads
(Bombina orientalis), five African
Dwarf Frogs (Hymenochirus boettgeri),
five Pacific Tree Frogs (Pseudacris
regilla), and two mudskipper species
(Periophthalmus
barbarous
and
novemradiatus) were obtained from the
Petco in Mission Viejo, the PetSmart in
Aliso Viejo, wild populations in Ladera
Ranch, and the Sandbar Pet Shop in
Mission Viejo, respectively. Each
species was divided into groups and
measured for their weight (grams) using
a OHAUS Scout Portable Electronic
Balance (ItinScale Company, Brooklyn,
New York, USA). Based upon the size
of the animal, we determined how much
dechlorinated water (pH = 6.86) to place
in the jars. In the capped containers,
Fire-bellied Toads and Pacific Tree
Frogs had 40mL, African Dwarf Frogs
and the small mudskipper (P.
novemradiatus) had 50 mL, and the
large mudskipper (P. barbarous) had
450 mL. These volumes allowed for the
animals to be almost completely
submerged while still providing them a
very small pocket of air to breath from
(less than 10 mL). Due to concerns of
asphyxiation, we chose to adjust the
Results
Rate of cutaneous oxygen
consumption was calculated for each
species using the data from Winkler’s
titration. The average rate of cutaneous
oxygen consumption was 3.1710-4
3.210-5 mg O2/min/g (SEM, n=5) for
Fire-bellied Toads, 1.4410-3 2.210-4
mg O2/min/g (SEM, n=5) for African
Dwarf Frogs, and 2.8910-3 5.410-4
mg O2/min/g (SEM, n=5) for Pacific
Tree Frogs. ANOVA and Bonferroni
post-hoc test revealed rate of cutaneous
O2 consumption of African Dwarf Frogs
is significantly higher than the other
amphibians. Rate of cutaneous O2
consumption for the mudskipper was
2.3410-3 mg O2/min/g (Figure 1).
Average O2 consumption rate (mg O2/min/g)
amount of time the animals were
submerged according to species. The
Fire-bellied Toads and Pacific Tree
Frogs were tested for ten minutes. The
African Dwarf Frogs and small
mudskipper were tested for twenty
minutes. The large mudskipper was
tested for thirty minutes. All tests were
conducted at room temperature (20.5°C).
To test the amount of dissolved oxygen
within the jars, we used LaMotte
Dissolved Oxygen Water Quality
Testing Kit (LaMotte Company,
Chestertown, Maryland, USA). After
removing the animals, eight drops of a
Manganous Sulfate Solution and eight
drop of Alkaline Potassium Iodide Aside
were added directly to the water samples
(40-50 mL). When this solution was
mixed, a precipitate formed and we had
to wait about thirty seconds to let it
settle. Eight drops of sulfuric acid
dissolved the precipitate and 20 mL were
put into a specialized test tube for the
titration. Eight drops of the Starch
Indicator turned the solution a dark blue
color. The titration was continued until
the solution was colorless. Upon reading
the syringe, we determined the dissolved
Oxygen ppm. These numbers and
weights were then calculated to specify
each
species’ cutaneous oxygen
consumption rate per gram on Excel.
In order to provide the study with
sufficient evidence, we obtained the
species’
metabolic
rate
through
concentration of CO2 as well .We placed
the animals into airtight containers
attach to a PASCO CO2 probe (PASCO
Scientific, Roseville, California, USA).
Each of the animals’ weights was
recorded before being placed in the airtight container for twenty minutes. This
data was used in the same formula to
calculate (on Excel) the average
metabolic rate for each species per gram.
0.004000
0.003000
0.002000
0.001000
0.000000
Figure 1. Average rate of oxygen consumption
for each species. Error bars indicate standard
error of mean.
Total rate of CO2 production
(rate of metabolism) was calculated for
all the species with the data from the
PASCO carbon dioxide probe. Average
rate of CO2 production was 1.54 0.15
mg CO2/min/g (SEM, n=5) for Firebellied Toads, 7.85 0.11 mg CO2/min/g
(SEM, n=5) for African Dwarf Frogs,
and 12.03 0.17 mg O2/min/g (SEM,
Average CO2 Production rate (mg
CO2/min/g)
16.00
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
Fire-bellied Pacific Tree African Mudskipper
Toads
Frogs
Dwarf Frogs
Figure 2. Average rate of carbon dioxide
production for each species. Error bars indicate
standard error of mean.
Percent Cutaneous Respiration
n=5) for Pacific Tree Frogs. Further
analysis by ANOVA and Bonferroni
post-hoc test showed rate of metabolism
of Fire-bellied Toads is significantly
lower than the other amphibian species.
Rate of CO2 production for the
mudskipper was 5.04 mg CO2/min/g
(Figure 2).
To determine the percent of
respiration that is done cutaneously, the
rate of cutaneous respiration (in moles
O2/min/g) was divided by rate of overall
respiration (in moles CO2/min/g) and
converted to percent. The average
percent cutaneous respiration was
2.8510-2 1.610-3 % (SEM, n=5) for
Fire-bellied Toads, 1.9010-2 1.910-3
% (SEM, n=5) for African Dwarf
Frogs, and 4.1410-2 2.710-3 %
(SEM, n=5) for Pacific Tree Frogs. The
African Dwarf Frogs have highest
percent cutaneous respiration and Pacific
Tree Frogs have the lowest. Statistical
analysis of data via ANOVA and
Bonferroni post-hoc test revealed
significant difference between all the
amphibian species. Percent cutaneous
respiration for the mudskipper was
6.8010-2 % (Figure 3).
0.0800
0.0700
0.0600
0.0500
0.0400
0.0300
0.0200
0.0100
0.0000
Fire-bellied Pacific Tree African Mudskipper
Toads
Frogs
Dwarf Frogs
Figure 3. Average percent cutaneous respiration
for each species. Error bars indicate standard
error of mean.
Discussion
According to the experiments the
African Dwarf frogs (Hymenochirus
boettgeri) had the highest cutaneous
respiration rate and higher overall
respiration rate than the Fire-bellied
Toads, the former is expected given their
aquatic existence while the latter is
understandable given their small body
size
and,
presumably,
higher
metabolism. They therefore proved to
be the model aquatic species we hoped
them to be. The Pacific Tree frogs’
(Pseudacris regilla) test results were not
what we expected. They were intended
to be our model terrestrial species, with
the low cutaneous respiration rate that
one would come to expect; however,
both their cutaneous and overall
respiration rates revealed not to be
significantly different from other
species. It is difficult to say why this
came to be; it is possible that P. regilla
is not the model terrestrial form we
thought it would be as the species does
favor moist areas and is primarily
nocturnal (both adaptations reduce
evaporative water loss and allow for a
higher cutaneous respiration rate), or
perhaps the equally curious Fire-bellied
Toad results are to blame for this
discrepancy. The Fire-bellied Toads
(Bombina orientalis) were intended to be
our semiaquatic species, with a
cutaneous respiration rate in between
that of the African Dwarf Frogs and
Pacific Tree frogs. The experiment did
not bear this out, however, with the
toads exhibiting lower overall respiration
rates than the other species. The reason
for this is unclear, it may be that their
warty skin texture and poison glands
inhibit cutaneous respiration to a degree,
or perhaps it is simply that B. orientalis,
being from a cooler climate than the
other species, naturally has a lower
overall metabolism.
In the case of the mudskipper’s
(Periophthalmus barbarus) results, as
only a single mudskipper specimen was
tested fully, no conclusion can be drawn.
Seeking to find another avenue
for congruencies in our data, we decided
to determine the ratio of cutaneous
respiration to overall respiration for each
species in the form of a percentage.
While the two data sets are not
measurements of the same gas or under
the same pressure (aquatic O2 vs. aerial
CO2), we believed that the ratio between
results could still be determined
accurately because the amount of O2
taken in during respiration is directly
proportional to the amount of CO2
expelled and because the differences due
to pressure and viscosity between
aquatic and aerial respiration were
constant for all tests, the end result being
that the ratios between the resultant data
would be consistent with each other,
which was all we needed. When the
calculations were performed, the result
was surprising: the percentage of
cutaneous respiration used by each
species relative to each other matched
our initial hypothesis. The African
Dwarf frogs were still in the aquatic
model, the Pacific Tree frogs had the
lowest ratio and therefore qualified as
the terrestrial model, and the Fire-bellied
Toads tested between the two other
amphibians thereby qualifying them as
the semiaquatic. If more mudskipper
specimens were tested and the
mudskipper’s percentage of cutaneous
respiration fell closest to that of the Firebellied Toads, then our hypothesis would
have been supported that mudskippers
have convergently evolved cutaneous
respiratory patterns similar to those of
amphibians that exhibit comparable
semiaquatic lifestyles. Our single
mudskipper’s cutaneous respiration
percentage is closest to that of the Firebellied Toads but no conclusion can be
drawn from it.
Though an exciting result, it must
be stressed that the hypothesis still
remains unconfirmed and needs to be
tested further with a wider array of
amphibian species and more mudskipper
specimens. Perhaps future tests could
also include other air-breathing fish,
such as polypterids, gars, anabantoids,
lungfish, leaping blennies, and perhaps
gilled amphibians and aquatic amphibian
larvae. It would be interesting to see if
any sort of linear result could be
achieved that matched evolutionary
pathways.
Acknowledgements
The authors would like to thank
Saddleback College Foundation and the
Biological Sciences Department of
Saddleback Community College for
supporting the project. We’d also want
thank professor Teh for lending his
expertise.
Authorship for this project was assigned
alphabetically.
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Review Form
Department of Biological Sciences
Saddleback College, Mission Viejo, CA 92692
Author (s):_Eidelstein, Perry, Saebi_______________________________
Title:_Comparison of Cutaneous Respiration Rates of Mudskippers and Amphibians
of Varied Ecology_
Summary
Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the
paper.
Cutaneous respiration is performed by all vertebrates but is greater in those living
in moist environments, such as the amphibian. Since terrestrial species don’t rely on
being submerged in water like the animals in moist environments, they developed
ways to prevent water loss instead. They then found a fish that performed
cutaneous respiration like an amphibian. This experiment wanted to test cutaneous
respiration by measuring o2 consumption and co2 production, and translating that
to a percentage of cutaneous respiration. They wanted to see if there was a
relationship in cutaneous respiration and evolution in species from water to land.
Results were not exactly what they wanted, some species exhibited respiration
qualities that qualified them as a model organism and others did not. More
experimentation on a larger and more variable scale needs to be done to really see
the correlation.
General Comments
Generally explain the paper’s strengths and weaknesses and whether they are serious, or important
to our current state of knowledge.
I thought the paper was very interesting and was trying to accomplish a lot by trying
to compare their results with the results expected from evolution. They introduced
the species well and reasons why those species were chosen. The discussion was
also great because it was easy to follow their results and the reasons they got them.
They also clearly explained how the results were relevant to their hypothesis and
the logic was understandable.
The paper should begin by explaining/defining cutaneous respiration so there’s
more background to it instead of just jumping into which species do it more in
comparison to others. Hypothesis should be more clearly stated as well – both
important for the reader. Also needs one more paper for citations.
There were also a few sentence formatting problems that should be addressed and
it wasn’t supposed to be in column form.
Technical Criticism
Review technical issues, organization and clarity. Provide a table of typographical errors,
grammatical errors, and minor textual problems. It's not the reviewer's job to copy Edit the paper,
mark the manuscript.
This paper was a final version
Recommendation
 This paper should be published as is
 This paper should be published with revision
 This paper should not be published

This paper was a rough draft