Chantal Eidelstein, Zachary Perry, and Azin Saebi

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 ray-finned 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 aOHAUS 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 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’cutaneousoxygen 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 air-tight 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.

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).

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 Fire-bellied 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, 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).

Figure 2. Average rate of carbon dioxide production for each species. Error bars indicate standard error of mean.

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 Fire-bellied 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 Fire-bellied 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.

Literature Cited

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Alderton, David. Encyclopedia of Aquarium & Pond Fish. N.p.: DK ADULT, 2008. Print.

Bartlett, R. D., Patricia Bartlett, and Billy Griswold. Reptiles, Amphibians, and Invertebrates: An Identification and Care Guide. 2nd ed. N.p.: Barron’s Educational Series, 2010. Print.

Gordon, M. S., Ng, W. W., & Yip, A. Y. (1978). Aspects of the physiology of terrestrial life in amphibious fishes. III. the chinese mudskipper periophthalmus cantonensis. The Journal of Experimental Biology, 72, 57-75. Retrieved from http://search.proquest.com/docview/83803184?accountid=14522