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Sex-related differences in metabolic rate and energy reserves in spring-breeding small-mouthed salamanders (Ambystoma texanum).

Michael S. Finkler and Kathryn A. Cullum
Indiana University Kokomo, Kokomo, IN 46904-9003, USA


We investigated potential differences in metabolism and energy substrate contents between male and females small-mouthed salamanders collected from breeding sites. Resting O2 consumption rates (VO2) of gravid females at 15 C was roughly double that males and post-gravid females. Whole-body triglyceride and glycogen contents were similar among males, gravid females, and post-gravid females, but free glucose contents were higher in gravid females than in males. Respiratory quotients for all groups were ~0.9, suggesting a relatively high dependence on carbohydrates to fuel metabolism during reproduction. These findings suggest that females have a considerably higher metabolic cost to reproduction than do males. Such differences may contribute to life history characteristics such as size dimorphism, differences in age of sexual maturity, asynchronous arrival times at breeding areas, and differential survival between the sexes.


Investigation into costs of reproduction has been a cornerstone of life history research, as it provides an excellent example of a trade-off (current reproductive expenditure vs. possible future reproductive output) that may influence the evolution of life history1. Potential costs of reproduction include allocation of energy for the synthesis of gametes, increased vulnerability to predation due to behavioral, morphological, and/or physiological changes, and increased energy expenditure for reproductive behaviors and general maintenance physiology2. Such costs may be particularly high for females, as larger amounts of energy are invested in the gametes2, and as increased costs for maintenance and activity3 and decreased locomotor performance4,5 often accompany gravidity.
Although considerable attention has been given to cost of reproduction in reptiles (most notably squamates), relatively little research into reproductive costs, particularly energetic costs, has been conducted on amphibians, and data for non-anuran taxa are especially lacking. For salamanders of the genus Ambystoma (Ambystomatidae), these energetic costs of reproduction may constitute a considerable encumbrance on the animals energy budget. Many northern species breed during the late winter or early spring, after a prolonged period of reduced feeding6. Moreover, the salamanders must often migrate appreciable distances from their overwintering sites to the breeding ponds6, thus introducing a potentially large transportation cost to the overall cost of reproduction.
Because female ambystomid salamanders produce much larger masses of gametes than do males, energetic costs of reproduction may be considerably higher in females than in male. Differences in the energetic cost of reproduction, therefore, may contribute to morphological, behavioral, and demographic differences between males and females. Female ambystomid salamanders are typically larger than male conspecifics6. Often, the two sexes arrive at breeding sites asynchronously, with males arriving sooner6. In addition, some studies have found a shift in demography from roughly equal numbers of males and females at metamorphosis to heavily male-biased sex ratios in adult populations, suggesting higher mortality in females than in males7,8.
The present study investigated potential differences in the energetic cost of reproduction between male and female small-mouthed salamanders (Ambystoma texanum). We hypothesized that reproductive females would have higher resting metabolic rates than would either male or post-reproductive females. We also hypothesized that females would have lower stored energy contents (fat and carbohydrates) than would males, and that post-reproductive females would have lower stored energy contents than would reproductive females.


Male (n = 17), gravid female (n = 10), and post-gravid female (n = 10) A. texanum were collected from breeding areas at Salamonie River State Forest, Wabash Co., IN, and Lost Bridge State Recreation Area, Huntington Co., IN during early March of 2000 and 2001. Animals were weighed within 16 hours of collection, and housed in plastic shoe boxes lined with moistened paper towels and maintained at 15 C on a 12h:12h light:dark cycle.
Resting oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured within 24 h of collection. Animals were placed individually into 250 ml sample

TABLE 1. Size measures of male, gravid female, and post-gravid female small-mouthed salamanders used in this study.

Group Live Mass (g) Carcass Mass (g) SVL (mm) Total Length (mm)

Males 7.4 1.8 (A) 7.4 1.8 (A) 76.5 5.4 (A) 130.4 9.5 (A)
Gravid Females 11.4 3.2 (B) 8.5 2.3 (A) 77.3 9.8 (A) 129.0 19.6 (A)
Post-Gravid Females 9.3 1.8 (AB) 9.4 1.9 (A) 82.6 4.1 (A) 141.4 10.9 (A)

* Data are presented as means SD, like letters in parentheses indicate no significant difference between groups (Tukey). Sample sizes: males n = 17, gravid females n = 10, post-gravid females n = 10.

bottles containing moistened paper towels to avoid dehydration of the subjects. Bottles were connected via plastic tubing to a Micro-Oxymax respirometry system (Columbus Instruments) then placed into a 15 C water bath. VO2 and VCO2 were measured automatically by the system at 2 h intervals over a 20-24 h period. Measurements for an individual were averaged to a single value prior to statistical analysis.
Following measurement of respiration, the animals were removed from their respirometry chambers and were anesthetized via submerged in a 0.67% MS-222 solution. The animals were then measured for snout-vent length and total length, female animals were dissected to remove any eggs present in the oviducts, and the animals were sacrificed by freezing. The carcasses were stored at 50 C until biochemical assays could be performed.
Carcasses were homogenized in 100 ml of distilled water. A 1 ml sample of the homogenate was then added to 2 ml of 0.6 N perchloric acid for deproteinization. After neutralization with 1 ml of 1 M KHCO3, the homogenate was analyzed for total triglyceride and free glucose contents using colorimetric techniques (Sigma 337 and 510). Glycogen content was determined by digesting a sample of the deproteinized and neutralized homogenate with amyloglucosidase and subtracting the free glucose content from the resultant total glucose content calculated. Total caloric contents were calculated based on published values for caloric yields from aerobic catabolism of free glucose, glycogen and triglycerides9.
Size data were analyzed using one-way analyses of variance (ANOVA), whereas respirometry and biochemical data were analyzed using analyses of covariance (ANCOVAs). In the ANCOVAs, Group (male, gravid female, or post-gravid female) was a fixed effect and carcass mass (i.e., without the eggs of gravid females) was the covariate. Initial analyses included interaction terms between the two effects, but as none of these interactions were significant, they were collapsed back into the model.


Size Measurements (Tables 1 and 2):
There was some evidence of size dimorphism in the salamanders used in this experiment. In analyses of size differences among the three groups, gravid females were more massive than either males or post-gravid females, but there were no significant differences in carcass mass, snout-vent length, or total length. When gravid and post-gravid females were combined,
TABLE 2. ANOVAs and ANCOVAs of size measurements, resting metabolism, and energy substrate levels in male, gravid female, and post-gravid female small-mouthed salamanders.

Group (fixed effect) Carcass Mass (covariate)
Parameter F df P F df P

Live Mass 10.06 2, 34 0.0004 -- -- --
Carcass Mass 3.08 2, 34 0.06 -- -- --
Snout-Vent Length 2.87 2, 34 0.07 -- -- --
Total Length 2.79 2, 34 0.08 -- -- --
Resting VO2 32.77 2, 30 < 0.0001 19.07 1, 30 0.0001
Resting VCO2 12.69 2, 13 0.0009 4.47 1, 13 0.054
Respiratory Quotient 0.50 2, 13 0.615 2.77 1, 13 0.120
Triglyceride Content 0.06 2, 32 0.938 2.79 1, 32 0.104
Free Glucose 3.41 2, 33 0.045 0.12 1, 33 0.727
Glycogen Content 0.47 2, 33 0.631 0.77 1, 33 0.386
Energy Content 0.01 2, 32 0.986 2.76 1, 33 0.107

however, the carcass mass of females (9.0 2.0 g) was also significantly greater than that of males (t = 12.87, df = 32, P < 0.0001), whereas neither snout-vent length (80.4 7.2) nor total length (136.3 15.8) differed significantly between the sexes.

Resting Metabolism (Tables 2 and 3):
Gravid females had markedly higher rates of O2 consumption and CO2 production than did either males or post-gravid females, with more than double the VO2 of post-gravid females.

TABLE 3. Resting oxygen consumption (VO2), resting carbon dioxide production (VCO2) and respiratory quotients (RQ) of male, gravid female, and post-gravid female small-mouthed salamanders at 15 C.

Group VO2 (l/h)*1 VCO2 (l/h)*2 RQ*2

Males 408.5 28.8 (A) 401.4 62.4 (A) 0.88 0.03 (A)
Gravid Females 790.3 43.0 (B) 752.3 53.2 (B) 0.91 0.03 (A)
Post-Gravid Females 386.9 37.9 (A) 399.1 63.5 (A) 0.91 0.03 (A)

* Data are presented as least-squares means SEM; like letters in parentheses indicate no significant difference between groups (P < 0.017, Bonferroni test)
1 Based on 2000 and 2001 data combined. Sample sizes: males n = 17, gravid females n = 10, post-gravid females n = 10.
2 Based on 2001 data only. Sample sizes: males n = 5, gravid females n = 7, post-gravid females n = 5.
TABLE 4. Whole body energy substrate contents for male, gravid female, and post-gravid female small-mouthed salamanders.

Group Triglyceride (mg)*1 Free Glucose (mg)*2 Glycogen (mg)*2 Stored Energy (cal)*1

Males 78.3 14.2 (A) 1.3 0.8
(A) 59.5 12.8 (A) 1005.3 165.4 (A)
Gravid Females 85.6 21.4 (A) 4.9 1.2
(B) 75.6 17.6 (A) 960.3 249.7 (A)
Post-Gravid Females 76.1 15.9 (A) 3.2 1.0 (AB) 53.7 14.7 (A) 965.9 185.0 (A)

* Data are presented as least-squares means SEM; like letters in parentheses indicate no significant difference between groups (P < 0.017, Bonferroni test).
1 Sample sizes: males n = 17, gravid females n = 9, post-gravid females n = 10.
2 Sample sizes: males n = 17, gravid females n = 10, post-gravid females n = 10.

In all three groups, however, respiratory quotients were approximately 0.9, suggesting a relatively high contribution of carbohydrates to overall metabolism.

Stored Energy (Tables 2 and 4):
We were unable to detect any differences among the three groups in terms of whole body triglyceride, glycogen, and total caloric contents. However, free glucose levels were significantly higher in gravid females than in males.


There was some evidence of sexual size dimorphism in the animals used in this study. Both snout-vent length and total length were slightly greater in females than in males (5.1% and 4.5% respectively). Differences between the sexes in mean linear measurements are similar to those reported for Ohio10 and Kansas11 populations of this species. In addition, carcass mass was significantly greater in females than in males, with mean values nearly 18% larger in females than in males.
Gravid females had resting metabolic rates that were roughly double those of males and post-gravid females. A similar elevation in the metabolic rate of gravid females compared to post-gravid females was reported for the lizard Sceloporus undulatus2, and likely constitutes a considerable component of the overall cost to reproduction. A variety of factors may contribute to this increase in metabolic rate, including proliferation and maintenance of the oviducts12, increased cost of locomotion13, and shifts in metabolic capacity14.
In both males and females, metabolism during the reproductive period appears to be fueled primarily through carbohydrate metabolism. In all three groups, respiratory quotients were approximately 0.9. Gravid females, which had the highest VO2, also had the highest free glucose contents. Although females generally had higher free glucose than did males, we were unable to detect a corresponding decrease in whole-body glycogen reserves in females relative to males. However, decreases in liver glycogen (the likely source of the free glucose) may have been obscured by variation in muscle glycogen.
The considerable elevation in metabolism by reproductive females compared to males may have important consequences on the life history. First, elevated resting metabolism may reduce the maximum sustainable level of activity for females, which could increase the duration of the migration from wintering site to breeding pool or necessitate that females delay migration until ideal environmental conditions (i.e., warm, wet weather) are available. This may, in part, account for regularly observed asynchronies in the arrival of males and females at breeding pools6,10. Second, the increased energy expenditure by females during reproduction could impact both growth and survival. Energy that could be used for growth is diverted to reproduction, which may slow growth rates in females compared to males and/or may necessitate greater foraging activity, thus increasing potential exposure to predators. Differential survivorship may account for the often male-biased sex ratios observed in breeding aggregations of ambystomids7. Third, the increased metabolic cost of reproduction in females may provide a driving force for the selection of larger female body sizes at reproductive maturity, thus inducing a delay in the age of first reproduction15 as well as sexual size dimorphism6,10,11.


1 Stearns, S. C. 1992. The evolution of life histories. Oxford: Oxford University Press.
2 Angilletta, M. J., and M. W. Sears. 2000. The metabolic cost of reproduction in an oviparous lizard. Funct. Ecol. 14:39-45.
3 Ryser, J. 1989. Weight loss, reproductive output, and the cost of reproduction in the common frog, Rana temporaria. Oecologia 78:264-268
4 Lee, S. J. M. S. Witter, I. C. Cuthill, and A. R. Goldsmith. 1996. Reduction in escape performance as a cost of reproduction in gravid startlings, Sturnus vulgaris. Proc. R. Soc. Lond. B 263: 619-624
5 Miles, D. B., B. Sinervo and W. A. Frankino. 2000. Reproductive burden, locomotor performance, and the cost of reproduction in free-ranging lizards. Evolution 54:1386-1395.
6 Petranka, J. W. 1998. Salamanders of the United States and Canada. Washington, D.C.: Smithsonian Institute Press.
7 Husting, E. L. 1965. Survival and breeding structure in a population of Ambystoma maculatum. Copeia 1965:352-362.
8 Sever, D. M. and C. F. Dineen. 1978. Reproductive ecology of the tiger salamander, Ambystoma tigrinum, in northern Indiana. Proc. Indiana Acad. Sci. 87:189-203.
9 Kleiber, M. 1961. The fire of life. New York: Wiley
10 Downs, F. L. 1989. Family Ambystomatidae. In: R. A. Pfingsten and F. L. Downs, eds. Salamanders of Ohio. Ohio Biological Survey Bull. 7:87-172.
11 Plummer, M. V. 1977. Observation on breeding migrations of Ambystoma texanum. Herpetol. Rev. 8:79-80.
12 Demarco, V., and L. J. Guillette, Jr. 1992. Physiological cost of reproduction in a viviparous lizard (Sceloporus jarrovi). J. Exp. Zool. 262:282-390.
13 Olson, M., R. Shine and E. Bak-Olsson. 2000. Locomotor impairment of gravid lizards: is the burden physical or physiological. J. Evol. Biol. 13:263-268.
14 Bauwens, D. and C. Thoen. 1981. Escape tactics and vulnerability to predation associated with reproduction in the lizard Lacerta vivipara. J. Anim. Ecol. 50:733-743.