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ABSTRACT
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.
INTRODUCTION
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.
METHODS
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.
RESULTS
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.
DISCUSSION
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.
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