Metamorphosis in Axolotl Salamanders: Study

The aim of the study was to explore the induction of metamorphosis in the Mexican axolotl salamander, Ambystoma mexicanum. The hypothesis for this experiment was that if the axolotls would undergo metamorphosis if exposed to thyroxine. Axolotls have been used in metamorphosis research studies because they do not naturally experience metamorphosis yet it can be induced, which can be controlled. Unlike axolotls, tiger salamanders spontaneously metamorphose in nature therefore making it much more difficult to control (Cano-Martinez, 1994). The axolotls were given an injection of thyroxine (1 mL of 5.0ug/g). Metamorphosis progressed and a reduction was seen in most of the neotenic characteristics of the experimental animals. The results were summarized in the graphs in Appendix B and the pictures in Appendix C. The graphs demonstrate a decreasing trend in the measurements. The pictures illustrate the results in the graphs, with very clear gill reduction and tail fin regression. The results from this study support the hypothesis that thyroxine would induce metamorphosis.

Introduction

Axolotl salamanders, Ambystoma mexicanum, are neotenic amphibians. The axolotl, as other neotenics, retains juvenile form into adulthood. Metamorphosis fails to occur and sexual maturity takes place in the larval state (Gilbert, 2000). Failure for axolotls to metamorphose is not related to a lack of thyroxine or triiodothyronine in the thyroid or a lack of thyroid stimulating hormone (TSH) in the pituitary but rather an inability for the pituitary to release TSH (Kuhn and Jacobs, 1989). Research with axolotls began in the 1860’s in France and a large proportion of the experimental stocks today can be traced to the initial research in France (Smith, 1989). Axolotls are used in metamorphosis research since their metamorphosis is easily induced and observed, hence the reason they were used in this experiment.

For this experiment, it was hypothesized that when A. mexicanum was exposed to thyroxine, it would undergo metamorphosis. On the other hand, the null hypothesis stated that when A. mexicanum was exposed to thyroxine, it would not undergo metamorphosis. If the axolotls were missing the hormone that normally would induce metamorphosis, thyroxine, then by exposing it to them would artificially stimulate metamorphosis. If the thyroxine induces metamorphosis, the dorsal ridge, gills, tail height, and tail fins were predicted to reduce in size and, given enough time, the dorsal ridge, gills, and tail fins would eventually disappear.

Materials and Methods

Fifteen axolotl salamanders were obtained from the Indiana University Axolotl Colony. The axolotls were housed in small plastic buckets with a mixture of dechlorinated water, NovAqua brand water conditioner, and Holtfreter salt (Fig. 1). The animals were fed liver meal pellets from the Indiana University Axolotl Colony twice a week, usually every Monday and Thursday. The water was changed on the same day and time as the animals were fed. Picture of the animals were taken on the same days as the feedings and water changings. The control and experimental animals were chosen at random by the students in the class. The control animals were 2, 3, 4, 9 and 14 whereas the experimental were 1, 5, 6, 7, 8, 10, 11, 12, 13, and 15.

Figure 1. Axolotl housing and setup.

The experimental axolotls were divided amongst the students so that every group had an axolotl. The controls were handled and measured by the same person every time. The procedure for measuring and weighing the axolotls followed the protocol of Cano-Martinez et al. (1994). The body weights were taken using a triple beam balance, and the other measurements were taken using a standard ruler or tape measure. The dorsal ridge length, gill length, tail height, lower tail fin height, upper tail fin height, and the body weights were taken once a week until the animals were dosed. Once the animals were dosed, the measurements and body weights were taken three times a week (Fig. 2).

Figure 2. Handling and measuring an axolotl salamander.

On 23 October 2002 the experimental animals (n=10) were given an introperitoneal injection (0.021mL of 2.5ug/g) of thyroxine and the control animals (n=5) were given the same amount of saline. On 7 November 2002 the control animals were given a 1 mL introperitoneal injection of saline. The experimental animas were given an introperitoneal injection (1 mL of 5.0ug/g) of thyroxine.

Results

As the semester progressed, the measurements and body weights remained relatively stable until the animals were given the second injection of thyroxine. The control animals did not experience any decrease in any of the measurements. The dorsal ridge length decreased for most of the experimental animals. For example, on the first day the length for animal 10 was 125 mm and on the last day it was 85 mm (Fig. 3).

Figure 3. Changes in Experimental Axolotl #10 – Sept. 12 (left), Nov. 21 (right).

Most of the experimental animals experienced some degree gill length reduction such as in animal 8 where the initial length on day one was 12 mm and on the last day was 0.5 mm (Fig. 4).

Figure 4. Changes in Experimental Axolotl #8 – Sept. 12 (left), Nov. 21 (right).

Reduction in the tail height, lower tail fin height, and upper tail fin height was experienced by most of the experimental animals. Examples are as follows: animal 1 had an initial tail height was 25 mm and the final height was 19, animal 6 initial had an lower tail fin height was 10 mm and the final height was 0 mm, and animal 11 had an initial upper tail fin height was 10 mm and the final height was 6 mm (see graphs). Animal 12 did not have a reduction in any of the measurements taken. The gill reduction is clearly evident in the picture for animal 8 (Fig. 4).

Discussion

By analysis of the graphs and pictures, the hypothesis that thyroxine induces metamorphosis can be reasonably accepted. The graphs demonstrate that after the thyroxine injections, regression in the various characters was marked. The pictures of animal 8, an experimental animal, compared to animal 3, a control animal, clearly demonstrate that the thyroxine induced metamorphosis. The gill reduction is the most marked change. With the exception of animal 12, the experimental animals displayed, to some degree, a reduction in the various characteristics. As expected, the control animals did not exhibit a reduction in any of the measurements taken, which is clearly demonstrated by the graphs and the pictures.

The initial dose of thyroxine did not induce metamorphosis possibly because the dose was not large enough compared to the body weight of the axolotls, or the dose was too small to which the animals would respond. Animal 12 did not undergo metamorphosis perhaps for a similar reason; however, it was sick before the experiment was begun. It is unknown whether or not that could have been a factor. Uncertainty in the measurements came from human error in not properly measuring the animals.

The results from this experiment suggest how development affects evolution. Heterochrony is the phenomenon wherein animals change in the relative time of appearance and rate of development of characters present in their ancestors (Gilbert, 2000). Neoteny is a type of heterochrony. Axolotls may give insight to either the path of evolution that amphibians took or a divergent path. If the gene(s) that control the expression of the various thyroid hormones are mapped and compared to other amphibians, path of evolution may be determined; whether the genes are missing or inhibited could give some thoughts to the scheme of evolution.

Conclusions

The body weights, dorsal ridge length, gill length, tail height, lower tail fin height, and upper tail fin height reduced in all but one of the experimental animals. The control animals did not experience any reduction. The results from this experiment indicate that thyroxine indeed does induce metamorphosis in Ambystoma mexicanum as demonstrated in Appendix B. One of the experimental animals did not metamorphose perhaps because the thyroxine dose was too small or the earlier illness prevented it. This serves as a model for vertebrate development and evolution because it demonstrates how metamorphosis proceeds and how early amphibians may have developed.

References Cited

Cano-Martinez, A., Vargas-Gonzalez, A., and Asai, M. 1994. Metamorphic stages in Ambystoma mexicanum. Axolotl Newsletter, 23:64-71.

Gilbert, S. F. 2000. Developmental Biology, 6^th edition. Sunderland, Massachusetts: Sinauer Associates, Inc., 749 pp.

Kuhn, E. R. and Jacobs, G. F. M. 1989. Metamorphosis; pp 187-197 in J. B. Armstrong and G. M. Malacinski (Eds.), Developmental Biology of the Axolotl. New York: Oxford University Press.

Smith, H. M. 1989. Discovery of the axolotl and its early history in biological research; pp.3-12 in J. B. Armstrong and G. M. Malacinski (Eds.), Developmental Biology of the Axolotl. New York: Oxford University Press.

Suggestions for Further Reading

Brown, D.D., The role of thyroid hormones in zebrafish and axolotl development. Proceedings of the National Academy of Science, USA v94. pp 13011-13016, Nov. 1997.

De Groef, B., Darras, V.M., Arackens, L., Gerets, H.H.J., Kuhn, E.R., and Geris, K.L., Changes of thyrotropin-releasing hormone levels in brain regions and pituitary during metamorphosis of Ambystoma mexicanum. Netherlands Journal of Zoology, 50(3): 343-354 (2000).

Denver, R. J. 1997. Proximate mechanisms of phenotypic plasticity in amphibian metamorphosis. American Zoologist, 37: 172-184.

Hanken, J., Jennings, D. H., and Olsson, J. 1997. Mechanistic basis of life-history evolution in anuran amphibians: direct development. American Zoologist, 37: 160-171.

Hayes, T.B. 1997. Steroids as potential modulators of thyroid hormone activity in anuran metamorphosis. American Zoologist. v37n2: 185-194.

Kupferberg, S.J., The role of larval diet in anuran metamorphosis. American Zoologist. v37n2: 146-159 (1997).

Stolow, M.A., Ishizuya-Oka, A., Su, Y. 1997. Gene regulation by thyroid hormone during amphibian metamorphosis: implications on the role of cell-cell and cell-extracellular matrix interactions. American Zoologist. v37n2: 195-207.

Wassersug, R.J. 1997. Where the tadpole meets the world-observations and speculations on biomechanical and biochemical factors that influence metamorphosis in anurans. American Zoologist. v37n2: 124-136

Wolffe, A.P., and Shi, Y. 1999 A hypothesis for the transcriptional control of amphibian metamorphosis by the thyroid hormone receptor. American Zoologist. 39:807-817.

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