THE JOURNAL OF EXPERIMENTAL ZOOLOGY 239:329-333 (1986)

New Function for the Granular Skin Glands of the Eastern Long- Toed Salamander, Ambystoma macrodactylum columbianum

THOMAS A. WILLIAMS AND JOHN H. LARSEN, JR. Department of Zoology and The Electron Microscopy Center, Washington State Uniuersity, Pullman, Washington 991644210

ABSTRACT While it has been accepted for over 50 years that salamanders use their tails for fat storage, no caudal adipose tissue was detected in the eastern long-toed salamander, Ambystoma macrodactylum columbianum. We provide evidence to show that a proteinaceous material, sequestered in the epidermally derived caudal granular skin glands, is the storage product. An increase in the size of these glands, at the expense of fat deposits in the tail, is interpreted as a solution to a conflict between two of the tail's major functions: nutrient storage and predator deterrence. Loss of caudal lipid allows for larger granular glands. With more secretory product available for defense, the possi- bility of the salamander's surviving an encounter with a predator is apparently enhanced. Storing protein is energetically beneficial because salamander diets are primarily protein, and it has been demonstrated by previous investigators that converting protein to fat for storage is metabolically ineficient.

A character is adaptive if it contributes to an organism's success within its biological niche (Bock, '80). Such characters may have several functions, which at times may be in conflict with one another. Therefore, compro- mises may be required if a structure has more than one function.

The salamander tail is an example of a complex structure that, because of its several functions, has apparently been involved in a number of these evolutionary "trade-offs." The tail of salamanders serves in nutrient storage (Noble, '31; Fitzpatrick, '71,'76; Maiorana, '75,'77) and in deterring predators (see Brodie, '77). During ambystomatid evo- lution, these activities apparently have been in conflict with one another. The antipreda- tor function of the tail is performed by secre- tions of epidermally derived granular glands. If, as has been assumed for over 50 years, large quantities of fat are stored in the tail (Noble, '31), then there is less space available in this structure for the synthesis and stor- age of a predator-repelling product. Since granular gland secretions of several species of Ambystoma, including A. macrodactylum, have been shown to be copious, toxic, and/or noxious (Brodie '77; Williams, '831, this does not seem to be the case.

We have examined the protectiodstorage problem using as our model the tail of the

salamander, Ambystoma macrodactylum col- umbianum. Here, for the first time, is pro- vided experimental evidence of nutrient storage in the tail of a terrestrial salaman- der; and we show that this material occurs in the granular skin glands as protein, an arrangement that resolves the apparent con- flict of function.

MATERIALS AND METHODS

A total of 24 male A. m. columbianum were collected in early spring, 1981, as they moved to the breeding ponds (Whitman Co., WA). While it is probable that any species of Am- bystoma would have provided a suitable study model, this subspecies was chosen be- cause it was readily available locally. Fe- males were not included in order to eliminate the possibility that energy demands of oogen- esis might confound the experimental re- sults. After capture, the salamanders were taken to the laboratory, where they were an- esthetized with a 2% aqueous solution of ether. Each animal was then uniquely toe- clipped for identification, and the following measurements were taken: snout-vent length (SVL), tail length ("I,), vertical and horizon- tal diameters (VD, HD) of the tail at a point 2 cm behind the posterior edge of vent.

Thomas A. Williams' present address is North Central Col- lege, 30 North Brainard Street, Naperville, IL 60566.

0 1986 ALAN R. LISS, INC.

330 T.A. WILLIAMS AND J.H. LARSEN, JR.

Three individuals were randomly placed in each of eight 19-cm glass finger bowls con- taining paper towels moistened with spring water. Two bowls were used for each of the following temperature and feeding treat- ments: 27°C fed, 27°C starved, 205°C fed, 20.5"C starved. All of the containers were exposed to light regime of 12L:12D for 60 days. Three times a week, those animals re- ceiving food were given as much beef liver and as many live earthworms as they would eat. Starved individuals were fed twice dur- ing the first 2 weeks to reduce the possibility of death by starvation as a result of an as- sumed poor posthibernation nutritional con- dition. During the 60-day experimental period, three of six starved-27°C animals died. The other salamanders (21) survived to the end of the experiment. After 60 days, the animals were anesthetized as described above, and final VD and HD measurements were made.

Changes in cross sectional area of the tail (ACSAT) were calculated from the initial and final VD and HD. The formula used was:

ACSAT = %VD x % H D x 7~

Values were then converted to percent change in order to reduce the effect of indi- vidual size differences. These changes were analyzed using a weighted two-way ANOVA.

Two fed and two starved A.m. colum- bianum were anesthetized and their tails ex- cised 2 cm behind the vent with a razor blade. For further comparison, we did the same to a salamander that had grown quite fat during 2 years of captivity. The latter was captured from the same population as the former. Two- cm sections of these tail pieces were fixed in Gilson's fluid and imbedded in paraffin ac-

cording to standard methods. From these preparations, transverse sections 10 pm thick were cut with a rotary microtome, affixed to glass slides, and stained with Mallory's con- nective tissue stain. Camera lucida drawings were made from representative sections of the five samples. From these drawings, areas of skeletal muscle and epidermal granular glands were quantified using a Numonics Model 1224 electronic digitizer.

Similar transverse sections from other in- dividuals were stained with Sudan Black II, Alcian blue-PAS, and ninhydrin-Schiff to stain for lipids, mucins and polysaccharides, and proteins respectively (Pearse, '68).

RESULTS

Tail diameter is dependent on the avail- ability of food (Table 1). While CSAT in- creased in fed animals, it decreased in starved individuals. Temperature difference had no statistically significant effect on CSAT.

Changes in tail diameter resulted from al- teration in the sizes of the epidermal granu- lar skin glands (Table 2, Fig. 1). While no determination of gland numbers was at- tempted, it appears that the increase in CSAT of fed animals resulted from hypertro- phy rather than hyperplasia. Measurements of tail cross section from two starved A.m. columbianum showed - 23% striated mus- cle, - 36%-granular epidermal gland, and -41% other tissue (primarily bone and con- nective tissue). While the tails of two fed salamanders contained - 19% striated mus- cle, -48% granular epidermal glands, and -33% other tissue, the tail of the 2-year captive had -8% striated muscle, -59% granular skin glands, and -33% other tis- sues. The CSAT of the 2-year captive was

TABLE 1. Changes in cross sectional area ofthe tail (CSAT) of21 adult Ambystoma macrodactylum columbianuma

Starved Starved Fed Fed warm cold warm cold

Average % change -20.78 (3) -20.70 (6) 17.38 (6) 37.78 (6) Standard deviation 10.00 15.80 21.84 25.23

Source sauares df sauare F Sum of Mean

Feeding (A) 2335.06 1 2335.06 29.33** Temperature (B) 103.24 1 103.24 1.27 ns AB interaction 104.84 1 104.83 1.32 ns Error 17 79.61

"Absolute changes in CSAT were converted to percentages to reduce the effect of animal size differences, and these percentage changes were analyzed using a weighted 2-way ANOVA. **denotes P < 0.01; ns denotes P > 0.05; sample size in parentheses.

SALAMANDER SKIN GLANDS 331

larger than that of any similar or larger sized experimental animals. Tests made for adi- pose tissue were negative for all tails used in this study (Fig. 1). A comparison of the amount of granular gland and striated mus- cle for the three salamander groups (Table 2) indicated that the gland regions are respon- sible for percentage alterations in CSAT.

The granular gland product stained posi- tively only for protein; there was no evidence of fat, mucus, or polysaccharides in these structures. No area of the tail stained posi- tively for lipid. While no histological differ- ences were evident between glands of starved and fed individuals, transmission electron microscopy revealed more secretory granules in glands of the latter (Williams, '83).

DISCUSSION

The CSAT of A.m. columbianum is clearly dependent on its feeding regime (Table 1). While food storage by the tail has been as- sumed by past investigators such as Noble ('311, the present study provides the first ex-

Starved Fed

TABLE 2. Differences in percentages ofgranular epidermal glands and skeletal muscle from two

representative starved, two representative fed, and a 2- year captiue A.m. columbianum

Treatment Captive Fed Starved

Average SVL (mm) 52.6 52.3 53.8 CSAT (mm') 16.84 11.70 6.33 Gland area (mm') 9.93 5.62 2.26 Gland area (%I 58.97 48.03 35.70 Muscle area (mm') 2.05 2.21 1.46 Muscle area (%I 8.21 18.89 23.06

perimental demonstration of this activity. Although tails have been cited as storage sites in Desmognathus (Fitzpatrick, '71,'76), Batrachoseps (Maiorana, '751, and Batrachu- perus (Reilly, '83), evidence has not been of- fered to show that the overall size of the tail varies with the amount of stored lipid. How- ever, the tail would seem to be an ideal place for food storage in salamanders. Since many salamanders spend much of the year in un- derground burrows, any increase in the di- ameter of the body would require the animal

Ca ptive

Fig. 1 Camera lucida drawings of representative cross sections of tails from 2 cm behind the vent of a starved, fed, and 2-year captive A.m columbianum Granular glands stippled; bar equals 1 mm.

332 T.A. WILLIAMS AND J.H. LARSEN, JR.

to occupy larger tunnels. By using the nar- rower tail for storing large amounts of en- ergy, this problem is eliminated.

Although Phisalix-Picot (1900,'22) pro- posed that amphibian granular glands evolved as nutrient storage structures, our histochemical tests provide the first demon- stration of the granular glands storing pro- tein. While the chemical nature of these ambystomatid glands has not been investi- gated, previously, peptides have been shown to be important constituents of other am- phibian toxins (Habermehl, '74), and the cur- rent study confirms the presence of stored protein in these structures for A.m. colum- bianum It is advantageous for salamanders to use protein for storage because they are almost exclusively carnivorous (Noble, '31). Since it has been determined that conversion of protein to fat for storage is metabolically inefficient for most animals (Mellward and Garlick, '76), the ability to store excess pro- tein appears to be energetically advanta- geous. Salamanders could reduce stored protein to amino acids and then reassemble them into other proteins or polypeptides as required.

As with many animals, A.m. columbianum will ingest more food, ad libitum, than it needs immediately. With an internal nu- trient storage site available, the salamander is able to store the surplus for future use (Noble, '31; Fitzpatrick, '76; Maiorana, '75,'77). Since the activity of A.m. colum- bianum is limited by environmental factors such as moisture and temperature, the abil- ity to store nutriment is of considerable adap- tive significance for these ectotherms.

We are not suggesting that protein is the only nutrient molecule stored by A.m. cob umbianum Salamanders do have lipids in the abdominal fat bodies, the liver, and cer- tain other areas of the body (Fitzpatrick, '71,'76). Also, some other species of the genus Ambystoma have caudal fat deposits (Wil- liams, '83). Changes in the girth of the abdo- men recorded during the course of this experiment were apparently due to altera- tions of body fat stores. In fact, storing both protein and fat would be highly useful to A. m columbianum, because individuals often face extended periods of fasting. Metaboliz- ing these two molecules would provide en- ergy, and, in addition, the tail-stored protein could serve as a source of nitrogen and essen- tial amino acids.

The gland apparently stores both toxin, (which may be a polypeptide), and protein in

a form that can be utilized as an energy source during starvation. (We have not estab- lished conclusively that the storage molecule and the toxin molecule are the same, al- though we suspect that they are, because a single product performing both functions could be more easily packaged and stored than two separate substances.)

While the most primitive members of the Ambystomatidae have the ability to deposit large amounts of fat in their tails, some of the more derived members of the family have larger caudal granular glands and smaller and fewer tail-fat deposits; the most ad- vanced species lack such fat deposits com- pletely (Williams, '83). The most parsi- monious interpretation of the data suggests that a reduction of fat deposits during phylo- geny coupled with increasingly larger caudal granular glands is an adaptation for both nutrient storage and predator repulsion. In the most advanced species of Ambystoma, the granular glands have assumed both func- tions, and fat is no longer stored in the tail (Williams, '83).

Secreting food stores to resist predation would appear to be debilitating but would be less severe than tail autotomy, the strategy employed by many salamander genera: Dee mognathus (Wake and Bresener, '66); Ensa- tina, Eurycea, Batrachoseps, Hemidactylum, Typhlotriton (Wake and Bresener, '66; Bro- die, '77); Pseudoeurycea, Bolitoglossa, and Chiropterotriton (Shaffer, '78); Thorius (Wake and Bresener, '66); and Chwglossa (Brodie, '77). In addition, DiGiovanni and Brodie ('81) and Williams ('83) demonstrated that stored secretory product in the caudal granular glands of Ambystoma is seldom, if ever, com- pletely depleted during an encounter with a predator. Ambystoma opacum individuals survived from 4 to 12 attacks from the short- tailed shrew (Blarina breuicaudata), a mam- mal known to feed readily on salamanders (Brodie et al., '79). Despite repeated attacks, these salamanders retained some gland prod- uct, thus reducing the likelihood of a lethal encounter. Also, these urodeles are burdened only with the synthesis of additional secere- tory material rather than the regeneration of an entire tail. It is unlikely that the loss of gland product by A.m. columbianum dur- ing an encounter with a predator would in- hibit reproduction to the same extent as does tail autotomy in I? larselli (Herrington and Larsen, in press) and B. attenuatus (Maior- ana, '77). Tail autotomy causes female B. attenuatus to forgo reproduction for a season

SALAMANDER SKIN GLANDS 333

and female l? larselli to resorb their matur- ing oocyte follicles.

Finally, it should be noted that caudal epi- dermal glands exhibiting a dual function (en- ergylstorage protection) are probably not unique to A . m columbianum Every species of Ambystoma examined has large numbers of granular glands in the tail (Williams, '83). While there is less evidence for this condition in plethodontid salamanders, Aneides fer- reus, Ensatina eschscholtzi Plethodon jor- dani, and l? glutinosus exhibit extensive concentrations of caudal granular gland fields and thus may have adaptations similar to those of Ambystoma.

ACKNOWLEDGMENTS

We would like to thank E. Brodie, J. Mal- latt, B. Shaffer, J. Thompson, and K. Tofte for their constructive comments on this project.

LITERATURE CITED

Arnold, S.J. (1982) A quantitative approach to antipre- dator performance: Salamander defense against snake attack. Copeia, 1982:247-253.

Bock, W.J. (1980) The definition and recognition of bio- logical adaptation. Amer. Zool., 20:217-227.

Brodie, E.D., Jr. (1977) Salamander antipredator pos- tures. Copeia, 1977.523-535.

Brodie, E.D., Jr., R.T. Nowak, and W.R. Harvey (1979) The effectiveness of antipredator secretions and behav- ior of selected salamanders against shrews. Copeia, 1979:270-274.

DiGiovanni, M., and E.D. Brodie, Jr. (1981) Efficiency of skin glands in protecting the salamander Ambystoma opacum from repeated attacks by the shrew Blurina breuicauda Herpetologica, 37:234-237.

Fitzpatrick, L.C. (1971) Influence of sex and reproductive condition on metabolic rates in the Allegheny Moun- tain salamander Desmognathus ochrophaeus. Comp. Biochem. Physiol., 40A:603-608.

Fitzpatrick, L.C. (1976) Life history patterns of storage and utilization of lipids for energy in amphibians. Amer. Zool., 16:725-732.

Habermehl, G.G. (1974) Chemical Zoology IX Amphibia and Reptilia. M. Florkin and B. Scheer, eds. Academic Press, New York, pp. 161-183.

Herrington, R.E., and J.H. Larsen (1986) Reproductive biology of the Larch Mountain salamander, Plethodon larselli J. Herpetol., in press.

Maiorana, V.C. (1975) Studies in the behavioral ecology of the plethodontid salamander Batrachoseps attenu- atus. Ph.D. Diss., University of California, Berkeley.

Maiorana, V.C. (1977) Tail autotomy, functional conflicts and their resolution by a salamander. Nature, 265533- 535.

Mellward, D.J., and P.J. Garlick (1976) The energy cost of growth. Proc. Nutri. SOC., 35339-349.

Noble, G.K. (1931) The biology of the Amphibia. Mc- Graw-Hill, New York, 577 pp.

Pearse, A.G.E. (1968) Histochemistry-Theoretical and Applied, 3rd ed, Vol. 1. Little Brown & Co., Boston, 759 pp.

Phisalix-Picot, M (1900) Recherches embryologiques, his- tologiques et physiologiques sur les glandes a venin de la salamandre terrestre. These de doctorat en m6de- cine. Paris, France.

Phisalix-Picot, M. (1922) Animaux Venimeaux et Venins, Vol. 2. Masson, France, 174 pp.

Reilly, S.M. (1983) The biology of the high altitude sala- mander Bastrachuperus mustersi from Afghanistan. Herpetol., 17:l-9.

Shaffer, H.B. (1978) Relative predation pressure on sala- manders (Caudata: Plethodontidae) along an altitudi- nal transect in Guatemala. Copeia, 1978:268-272.

Wake, D.B., and I.G. Bresener (1966) Functional mor- phology and evolution of tail autotomy in salaman- ders. J. Morph., 122;265-305.

Williams, T.A. (1983) Structure and functions of the granular skin glands of the salamanders of the family Ambystomatidae (Amphibia: Urodela). Ph.D. Diss., Washington State University, Pullman.