Hormones and Behavior 55 (2009) 24–32
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Hormones and Behavior
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y h b e h
The effects of maternal corticosterone levels on offspring behavior in fast- and
slow-growth garter snakes (Thamnophis elegans)
Kylie A. Robert 1, Carol Vleck, Anne M. Bronikowski ⁎
Department of Ecology, Evolution and Organismal Biology, Iowa State University, Ames, Iowa, USA
a r t i c l e
i n f o
Article history:
Received 7 January 2008
Revised 12 July 2008
Accepted 15 July 2008
Available online 29 July 2008
Keywords:
Stress
Corticosterone
Viviparity
Reptile
Transdermal application
Morphology
Locomotor performance
Escape behavior
a b s t r a c t
During embryonic development, viviparous offspring are exposed to maternally circulating hormones.
Maternal stress increases offspring exposure to corticosterone and this hormonal exposure has the potential
to influence developmental, morphological and behavioral traits of the resulting offspring. We treated
pregnant female garter snakes (Thamnophis elegans) with low levels of corticosterone after determining both
natural corticosterone levels in the field and pre-treatment levels upon arrival in the lab. Additional
measurements of plasma corticosterone were taken at days 1, 5, and 10 during the 10-day exposure, which
occurred during the last third of gestation (of 4-month gestation). These pregnant snakes were from replicate
populations of fast- and slow-growth ecotypes occurring in Northern California, with concomitant short and
long lifespans. Field corticosterone levels of pregnant females of the slow-growth ecotype were an order of
magnitude higher than fast-growth dams. In the laboratory, corticosterone levels increased over the 10 days
of corticosterone manipulation for animals of both ecotypes, and reached similar plateaus for both control
and treated dams. Despite similar plasma corticosterone levels in treated and control mothers,
corticosterone-treated dams produced more stillborn offspring and exhibited higher total reproductive
failure than control dams. At one month of age, offspring from fast-growth females had higher plasma
corticosterone levels than offspring from slow-growth females, which is opposite the maternal pattern.
Offspring from corticosterone-treated mothers, although unaffected in their slither speed, exhibited changes
in escape behaviors and morphology that were dependent upon maternal ecotype. Offspring from
corticosterone-treated fast-growth females exhibited less anti-predator reversal behavior; offspring from
corticosterone-treated slow-growth females exhibited less anti-predator tail lashing behavior.
© 2008 Elsevier Inc. All rights reserved.
Introduction
Glucocorticoid hormones are released from the adrenal cortex in
response to activation of the hypothalamic–pituitary–adrenal (HPA)
axis in mammals and hypothalamic–pituitary–interrenal (HPI) axis in
reptiles (Norris and Jones, 1987; Greenberg and Wingfield, 1987;
McEwen and Wingfield, 2003). Individual glucocorticoid modulation
can vary within populations and between individuals in response to
body condition, reproductive state, age, sex, disease status, and social
status (Wingfield et al., 1992; Woodley and Moore, 2002). In addition,
external environmental conditions such as temperature, rainfall, and
food availability can also elicit variation in rate, duration and
magnitude of glucocorticoid response (Dufty et al., 2002; Romero,
2002). Reptiles are ideal model organisms for studies of the
⁎ Corresponding author. 253 Bessey Hall, Iowa State University, Ames, Iowa 50011,
USA. Fax: +1 515 294 1337.
E-mail addresses: [email protected] (K.A. Robert), [email protected]
(A.M. Bronikowski).
1
Present address: FNAS School of Animal Biology, University of Western Australia,
Perth, Australia.
0018-506X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.yhbeh.2008.07.008
glucocorticoids and stress. Most reptiles respond to stressors by
increasing plasma levels of corticosterone (Greenberg and Wingfield,
1987; Guillette et al., 1995; Lance, 1990). Corticosterone is the major
glucocorticoid in reptiles (Greenberg and Wingfield, 1987) and it can
be experimentally manipulated by non-invasive external application
(Knapp and Moore, 1997; Meylan et al., 2002).
External stressors are thought to impact reproduction negatively,
beyond the natural stress that gravidity imposes on a female. This
concept, that life-history related stress and unpredictable external
stress may require differential modulation of the HPA/HPI axis, has
been termed allostasis (reviewed in Landys et al., 2006). Furthermore,
it is increasingly clear that conditions experienced during embryonic
development can influence offspring morphology and behavior after
parturition (Clark and Galef, 1998; Mousseau and Fox, 1998; Painter et
al. 2002). Maternal effects can be mechanisms that pre-adapt
offspring to external environmental conditions. Specifically, corticosterone can act as a cue for developing offspring, providing information
on maternal and environmental condition (Dufty et al., 2002). Given
that hormones of the HPA/HPI axis can have vast influences on
embryonic development and survival, it has been hypothesized that
females may evolve mechanisms to reduce the corticosterone
K.A. Robert et al. / Hormones and Behavior 55 (2009) 24–32
Table 1
Life history and habitat differences for the two ecotypes of garter snake (summarized
from Kephart and Arnold, 1982, Bronikowski and Arnold, 1999, and Bronikowski
unpublished)
Lakeshore — fast growth
Meadow — slow growth
Life history
Grow quickly to a larger adult size
(450–700 mm SVL)
Early reproduction (1st litter at 3 years
of age)
Low annual probability of survival
(adults = 0.46)
Median lifespan = 4 years
Grow slowly to a smaller adult size
(400–55 mm SVL)
Late reproduction (1st litter at 5–7 years
of age)
High annual probability of survival
(adults = 0.75)
Median lifespan = 8 years
Habitat
Constant (fish) prey and water availability Ephemeral (frog) prey and water
availability
High avian predation pressure
Low avian predation pressure
Warmer (average = 25 °C) nighttime
Cooler (average = 20 °C) nighttime
temperature
temperature
response to stressors during pregnancy to protect their developing
embryos from harmful effects of increased HPA/HPI activity (Cree et
al., 2003). Such a lowered response is seen during pregnancy in rats
(Neumann et al., 1998) and a change in adrenal sensitivity at different
stages of pregnancy is seen in mice (Barlow et al., 1976).
In many species, variation in locomotion abilities is likely to have
important fitness consequences (Meylan and Clobert, 2004; Miles,
2004; Husak et al., 2006) because many key behaviors involve
locomotion (e.g., foraging, predation escape, dispersal). Corticosterone
elevation may enhance locomotor performance in offspring through
changes in energy mobilization, as has been demonstrated in birds
(Belthoff and Dufty, 1995) and lizards (Miles et al., 2007), or
alternatively cause a reduction in locomotor performance due to
deleterious changes in offspring morphology (e.g., smaller body sizes,
worse body condition). Locomotor performance is widely used as a
measure of fitness in reptiles (Christian and Tracy, 1981; Arnold, 1983;
Jayne and Bennett, 1990; Downes and Shine, 1999; Garland, 1999). For
example, locomotor performance in the common garter snake
(Thamnophis sirtalis) is positively correlated with survival; faster
offspring are more likely to survive to their second year than slower
offspring (Jayne and Bennett, 1990). Locomotor performance is also
positively correlated with survival under natural conditions in several
squamate lizards (Warner and Andrews, 2002; Miles, 2004) and
turtles (Janzen, 1995) and correlates with lifespan in colubrid snakes
(Robert et al. 2007). Locomotor capacity is a highly repeatable
measure over time; an individual who is relatively fast at one time
will remain relatively fast at another. To date all species examined
have shown high repeatability of locomotor performance (Brodie,
1989; Brodie and Russell, 1999; reviewed in Bennett and Huey, 1990).
This is important for measuring performance of neonates and
inferring future lifetime effects.
Locomotor performance varies among individuals in nature
(Bennett and Huey, 1990; Garland and Losos, 1994; Aerts et al.,
2000; Irschick and Garland, 2001). It is a trait that is easily evolved in
laboratory selection experiments (Bronikowski et al., 2001; Garland
and Freeman, 2005; Rezende et al., 2006), and in natural experiments
in the field (e.g., Reznick et al., 2004). In addition to locomotion,
colubrid snakes have several supplementary behaviors that can be
classified as anti-predation behavior. For example, individuals can
flee; remain still (crypsis); display warnings and diversions such as tail
lashing, mouth gaping and skin color flushing; expel cloacal secretions; rear up; flatten; and strike (reviewed in Ford, 1996; Robert et al.,
2007).
Our study focuses on how maternal corticosterone levels during
pregnancy affect offspring morphology, locomotor performance and
anti-predator behaviors in two ecotypes of the western terrestrial
25
garter snake (Thamnophis elegans). These ecotypes, found in Lassen
County, California, exhibit two distinct life-history strategies (Bronikowski and Arnold, 1999). Snakes from lakeshore sites grow fast,
mature early and die young (ca. 4 years of age). Snakes from mountain
meadow sites grow slowly, mature later in life, and live twice as long
(ca. 8 years) (Table 1). Lakeshore/fast-growth [“L/fast”] snakes invest
heavily in growth and reproduction, whereas meadow/slow-growth
[“M/slow”] snakes invest heavily in maintenance and survival (Sparkman et al., 2007). Previous work indicates that these differing lifehistory traits are under both genetic and environmental control
(Bronikowski and Arnold, 1999; Bronikowski, 2000). As well, populations of the two ecotypes have limited gene flow and are differentiated
based on neutral molecular markers (Manier and Arnold, 2006;
Manier et al., 2007).
Materials and methods
This project was approved by the Iowa State University Institutional Animal Care and Use Committee (log number: 3-2-5125-J and
8-06-6198-J).
Study animals
The western terrestrial garter snake (T. elegans) is a medium sized
diurnal colubrid snake (adult snout-vent length [SVL]: 300–500 mm,
juvenile SVL: 100–200 mm), and is widely distributed throughout
western North America. The focus of our study is on replicate
populations (3 per ecotype) in close proximity (5–10 km) in the
vicinity of Eagle Lake, Lassen County, California. They occur in two
primary habitats (lakeshore and meadow) with associated life-history
patterns of fast growth/short life and slow growth/long life (Bronikowski and Arnold, 1999).
Throughout June 2006 we collected 68 pregnant females, 35 L/fast
and 33 M/slow, and returned them to the laboratory until parturition
(August/September). Pregnant females are easily identified by palpation of their abdomen for embryos. At the time of hand-capture a
blood sample was drawn within 3 min of capture (to establish baseline
field measures of corticosterone) and females were weighed and
measured. We collected snakes during the morning and randomized
morning collections to control for this potential source of variation.
We verified that time of morning was not a significant explanatory
Fig. 1. Curves of percent bound I125 tracer (%B/Bo) against log-transformed serial dilutions
of pooled L/fast (solid diamonds, 1:5–1:160) and M/slow (open squares, 1:5–1:160) plasma
samples. Corticosterone standards (shaded circles, 12.5–1000 ng/ml). Serial plasma
dilutions were parallel to the standard corticosterone curve (t =3.623, P = 0.171) and a
plasma dilution of 1:40 displaced between 50–60% of the I125 labeled hormone from the
antibody.
26
K.A. Robert et al. / Hormones and Behavior 55 (2009) 24–32
Blood sampling
Table 2
Analyses of variance of maternal traits
Dependent variable
F(df1, df2)
Source of variation
Dam length
Ecotype
Treatment
Ecotype × treatment
Population (ecotype)
Time
Time × treatment
Time × ecotype
Time × ecotype × treatment
Repeated cort
Field cort
Reproductive failure
0.28 (1, 59)
0.13 (1, 59)
0.10 (1, 59)
0.14 (1, 59)
1.04 (4, 59)
25.68 (3, 181)⁎⁎
2.69 (3, 181)
1.48 (3, 181)
1.46 (3, 181)
0.72 (1, 56)
5.10 (1, 56)⁎
0.27 (1, 56)
0.83 (1, 56)
0.96 (4, 56)
1.18 (1, 59)
0.92 (1, 59)
4.52 (1, 59)⁎
0.32 (1, 59)
1.04 (4, 59)
“Repeated cort" is four measurements of corticosterone in pregnant females (arrival, and
days 1, 5, and 10) of corticosterone application). “Field cort" is level at capture in nature.
“Reproductive failure" includes stillborns, undeveloped yolks and deaths within the first
month. See text for details.
⁎ P b 0.05,
⁎⁎ P b 0.0001.
Blood samples (100–200 μl whole blood) were drawn from the
ventral coccygeal (tail) vein using heparinized 1 ml insulin syringes
within 3 min of handling in the mornings (9:00 AM ± 1 h) (for
consistency with field-collected samples). Blood samples for the
experimental females were repeated measures taken at: field capture,
6 days post arrival, and days 1, 5, and 10 of the 10-day exposure. Whole
blood was centrifuged and plasma stored at −80 °C until assayed.
Blood samples not obtained within 3 min, incorrectly handled, or if of
insufficient volume were excluded (three L/fast field samples).
A measure of offspring corticosterone was assessed from a pooled
blood sample across all litter mates in each litter, taken at 4 weeks
(±1 week) of age. Pooling was necessary within each litter to obtain
the required volume of plasma (10–20 μl whole blood was collected
per baby). One L/fast litter and three M/slow litters yielded insufficient
plasma volume for the corticosterone assay.
Hormone assays
variable prior to subsequent statistical analyses. On return to the
laboratory pregnant females were housed individually in glass aquaria
(26 mm W × 50 mm L × 30 mm H) with paper substrate and water
dishes with hollowed rims that doubled as shelter sites. Dams were
fed twice weekly on a diet of live goldfish (3–4 per feeding), provided
heating (thermal gradient 24–34 °C) and lights on a 12 h: 12 h light:
dark cycle. Females from both ecotypes were randomly assigned to
one of three treatment groups: control (no treatment) N = 20 [10 L/fast
and 10 M/slow], placebo (sesame oil) treatment N = 19 [10 L/fast and 9
M/slow], or corticosterone-treated N = 29 [15 L/fast and 14 M/slow]).
Following birth, females were returned to the field and released at
their point of capture.
Hormonal treatment
We manipulated circulating levels of corticosterone using a noninvasive method (Meylan et al., 2002) based on a modification from
Knapp and Moore (1997). Corticosterone was delivered transdermally
to the snakes using a mixture of the steroid hormone and sesame oil.
Corticosterone dosage was calculated based on body mass at capture
so that treated individuals received 0.55 μg/g mass. We diluted
corticosterone (Sigma C2505 [346.47 FW] 92%) in commercial pure
sesame oil (3 μg/μl sesame oil). Previous studies in the common lizard
(Lacerta vivipara) (Meylan et al., 2002; Meylan and Clobert, 2004,
2005) and the tree lizard (Urosaurus ornatus) (Knapp and Moore, 1997)
applied 4.5 μl of the same concentration per animal (13.5 μg
corticosterone/lizard). The average size for both lizard species is
around 5 g; therefore both studies used a corticosterone dosage of
∼ 2.7 μg/g body mass to induce a 13–14 fold increase in baseline
plasma corticosterone levels. Therefore using 0.55 μg/g in this study,
we predicted a 3–4 fold increase in baseline corticosterone levels,
chosen to ensure that the increase was within the natural range rather
than a pharmacological range, which would probably induce mothers
to abort their pregnancies.
Following acclimation to lab conditions for six days, a pretreatment (arrival) blood sample was drawn at 9 am (±30 min) within
3 min of removal from their home cage to gain a laboratory baseline
corticosterone level. Treatments were initiated 5 days following this
initial bleed by applying either nothing, sesame oil alone, or sesame oil
corticosterone mix to the dorsal skin surface daily for 10 days. We
treated the snakes each evening immediately prior to lights off
because they were less active in the evenings, which allowed for
absorption across the skin surface. The high concentration of lipids in
reptile skin enables lipophilic molecules, such as steroids to readily
cross the skin surface (Mason, 1992).
Total plasma corticosterone was assayed using a double antibody
corticosterone radioimmunoassay kit (Catalog # 07-120103, MP
Biomedical, Orangeburg, NY). In brief, we followed the MP Biomedical
protocol for the I125 corticosterone RIA, with a few exceptions. We
quartered the volume of all reagents, diluted the 25 ng/ml standard
1:2 with steroid diluent to produce a 12.5 ng/ml standard, and altered
the plasma dilution for the sample unknowns following assay
validation (the dilution that provided the closest to 50% bound was
determined to be the ideal dilution for experimental assays).
To validate plasma corticosterone RIA, we tested for parallelism
with the standard dilutions in pooled plasma samples (five pooled
individual plasma samples from extra M/slow snakes and five pooled
plasma samples from extra L/fast snakes). Assay precision was
assessed by calculating intra- and inter-assay coefficients of variation
(CVs) of the percentage bound of the internal controls. Our two
pooled samples and the kit-provided low control were used as
internal controls. Randomly assigned groups of samples were
assayed back-to-back within a 48-hour time period using the same
reagents in order to reduce inter-assay variability (N = 4 assays).
Variations among replicates within an assay were used to calculate
Fig. 2. Least-square means with 1 SE for plasma corticosterone [CORT] (ng/ml). (A)
Pregnant females at time of field capture. Meadow/slow females have significantly
higher baseline corticosterone than L/fast females at the time of capture (F1,56 = 5.10,
P b 0.05). (B) Pooled offspring per litter at 1 month of age. L/fast offspring have
significantly higher corticosterone levels than M/slow offspring at 1 month of age
(F1,56 = 4.40, P b 0.05). This was true irrespective of corticosterone treatment (effect of
treatment and ecotype ⁎ treatment interaction P N 0.05). The different maternal treatments are untreated (white bars) and CORT treated (black bars).
K.A. Robert et al. / Hormones and Behavior 55 (2009) 24–32
27
given a unique number (marked with permanent markers) and then
housed in litter groups with water provided ad libitum. For each dam,
we computed reproductive failure as the numerical proportion of her
litter represented by stillbirths, yolks, and offspring deaths in the first
month of life. Overall, the 68 dams gave birth to litters that ranged
from 3–17 live offspring. Neonates are born with yolk reserves and
were not fed prior to the behavior tests.
Offspring locomotor performance and behavior
Fig. 3. Least-square means from repeated measures analysis of variance of plasma
corticosterone (CORT) levels (ng/ml). Values of all four groups, M/slow-CORT, M/slownoCORT, L/fast-CORT and L/fast-noCORT, increased over the sampling duration:
field → arrival → day 1. The four curves do not differ from each other in their shape
(ecotype × treatment × time effect, P N 0.05). Field values are indicated on the graph but
were not included in the repeated measures analysis.
intra-assay variation; average values across assays were used to
calculate inter-assay variation. The assay uses a specific rabbit
anticorticosterone antibody, and has a sensitivity of 7.7 ng/ml and
the following cross-reactivity at 50% displacement compared to
corticosterone 100.00%: Desoxycorticosterone 0.34%, Testosterone
0.10%, Cortisol 0.05%, Aldosterone 0.03%, Progesterone 0.02%,
Androstenedione 0.01%, all others b0.01%.
Serial dilutions (1:5, 1:10, 1:20, 1:40, 1:80, 1:160) of five pooled
plasma samples from L/fast and M/slow ecotypes yielded displacement curves that were parallel to the standard corticosterone curve
(t = 3.623, df = 1, P = 0.171) (Fig. 1). A dilution of 1:40 displaced between
50 and 60% of the I125-labeled hormone from the antibody while
maintaining 100% accuracy for the kit-provided control. All plasma
samples were diluted 1:40 (e.g., 5 μl of plasma in 195 μl steroid
diluent) for assays. Intra-assay variation was 4.7% for the low control
and 5.5% for the reference sample. Inter-assay variation was 9.1% for
the low control and 11.3% for the reference sample.
Offspring morphology
On the day of birth offspring were removed from their mother's
cage, sexed, weighed (g), measured (snout-vent length [SVL], mm) and
All measurements were made at 1 month of age at 28 °C (preferred
body temperature range 28–32 °C, Huey et al., 1989; Arnold and
Peterson, 2002) on a linear racetrack measuring 4 cm wide and 120 cm
in total length. Photocells located at 25 cm intervals along the
racetrack recorded the cumulative time taken for snakes to cross each
successive infrared beam. The surface of the track consisted of rough
sand paper to facilitate locomotion. For offspring locomotor performance, each snake was run three times with a 20 min break between
runs. To begin a trial, an individual was transferred directly from its
container to the holding area of the racetrack (first 10 cm prior to first
photocell), whereupon it was released and allowed to traverse a 1 m
distance; if necessary, it was chased with a paintbrush with light taps
to the tail. Sprint speed was calculated as the average 1 meter speed
over the three trials. Burst speed was the maximum 25 cm speed over
all three trials. We also recorded escape behavior as individuals that
stopped, reversed (turned back) and then continued. Any individual
that could not locomote due to spinal deformity was excluded from
trials (12 individuals: five control and 7 corticosterone-treated, from a
total of 8 litters). Defensive tail lashing/rattles were also recorded. No
individual displayed other aggressive or anti-predatory behaviors
during locomotor trials, such as mouth gapes or striking.
Statistical analysis
Statistical analyses were performed using the mixed-model
procedure (Proc Mixed) in SAS software (SAS 9.1.3, SAS Institute Inc.,
Cary, NC). There were eight dependent variables. The three dependent
variables for dams were: field plasma corticosterone, reproductive
failure, and four repeated measures of laboratory plasma corticosterone (6 days post arrival to laboratory “arrival,” day 1 of 10-day
treatment initiated five days after the “arrival” sample was taken, day
5 of the 10-day treatment, and day 10 – the final day – of
corticosterone, placebo, or control treatment). The five dependent
variables for offspring were: birth mass, birth SVL, sprint speed at
1 month of age, burst speed at 1 month of age, and plasma
corticosterone from plasma pooled for each litter at 1 month of age.
The general linear models are stated below. For all analyses, placebo
(treated with oil) and control females (“treated” with empty pipette
tip) did not differ from each other (P N 0.05 in all tests, data not
Table 3
Mixed model analysis of variance for offspring traits (morphology, performance, and corticosterone)
Dependent variable
F(df1, df2)
Source of variation
Sex
Ecotype
Treatment
Ecotype × treatment
Ecotype × sex
Treatment × sex
Ecotype × treatment × sex
Pop (ecotype)
Morphology
Performance
Body length (SVL (mm))
Body mass (g)
Sprint speed (SVL/s)
13.5 (1, 428)⁎⁎⁎
17.9 (1, 58)⁎⁎⁎
2.74 (1, 428)
0.99 (1, 428)
1.87 (1, 428)
9.57 (1, 428)⁎
2.61 (1, 428)
1.44 (4, 58)
2.03 (1, 428)
4.67 (1, 58)⁎
0.40 (1, 428)
0.86 (1, 428)
1.73 (1, 420)
0.86 (1, 428)
1.55 (1, 428)
1.12 (4, 58)
12.0
2.49
0.31
0.57
0.54
0.59
0.56
4.57
(1, 428)⁎⁎
(1, 58)
(1, 428)
(1, 428)
(1, 428)
(1, 428)
(1,428)
(4, 58)⁎
Litter nested within population is a random effect; thus ecotype and population (ecotype) are tested over the litter mean squares.
⁎ P b 0.05,
⁎⁎ P b 0.001,
⁎⁎⁎ P b 0.0001.
Corticosterone
Burst speed (SVL/s)
Offspring cort
1.37 (1, 428)
0.52 (1, 58)
0.24 (1, 428)
0.47 (1, 428)
1.73 (1, 428)
0.13 (1, 428)
0.24 (1, 428)
1.11 (4, 58)
NA
4.40 (1, 56)⁎
0.46 (1, 56)
0.48 (1, 56)
NA
NA
NA
1.02 (4, 56)
28
K.A. Robert et al. / Hormones and Behavior 55 (2009) 24–32
shown). Therefore, control and placebo animals were grouped
together in a “no Corticosterone” treatment group for the analyses
based on this lack of significant difference (with α = 0.05, β (power) =
0.77 based on the observed variability in field corticosterone measures;
it is possible that we failed to detect a true difference between placebo
and control animals).
The general linear model (GLM) for field plasma corticosterone and
for reproductive failure was:
Response variable ¼ μ þ Trt þ E þ Trt⁎E þ PopðEÞ þ SVL þ e
The GLM for the four repeated measures of dam plasma corticosterone
measured in the laboratory was:
Repeated measures ¼ μ þ Trt þ E þ Trt⁎E þ PopðEÞ þ Time þ Time⁎E
þ Time⁎Trt þ Time⁎Trt⁎E þ SVL þ e
The GLM for the offspring plasma corticosterone, pooled for each
litter, was:
Response variable ¼ μ þ Trt þ E þ Trt⁎E þ PopðEÞ þ e
The GLM for each of: birth mass, birth SVL, 1-month sprint speed and
1-month burst speed was:
¼ μ þ Sex þ Trt þ E þ Sex⁎Trt þ Sex⁎E þ Trt⁎E þ Sex⁎Trt⁎E þ PopðEÞ
þ LitterðPopðEÞÞ þ e
where:
Trt is corticosterone treatment: Corticosterone vs. no Corticosterone;
E is ecotype: L/fast- vs. M/slow-growth ecotype; Pop(E) is population
nested within ecotype (three L/fast populations and three M/slow
populations); Sex is male vs. female; and Litter is random litter nested
within population.
Reversal and tail lashing behavior were each analyzed using the Gstatistic test of independence (Sokal and Rohlf, 1981) testing offspring
from untreated dams versus corticosterone-treated dams within
ecotype. Test of slope heterogeneity was used to determine if curves
of serially diluted pooled plasma samples from each ecotype of snakes
were parallel to log-transformed corticosterone standard curves
(Neter et al., 1990).
Reproductive success
Corticosterone-treated females from both ecotypes were more likely
to produce stillborn offspring, and suffer from higher total reproductive
failure (stillborn, deformities, arrested development and neonate death
before one month of age). Least-square means for corticosterone-treated
percent failure=28.7 ±5.2%, n =29; untreated =13.8 ±4.8%, n =39 (Table 2).
Offspring morphology
From 68 females 511 lab born offspring were produced (untreated —
L/fast: 20 litters and 213 offspring, M/slow: 19 litters and 99 offspring;
CORT treated — L/fast: 15 litters and 133 offspring, M/slow: 14 litters and
66 offspring) (Table 3). Both ecotypes (ANOVA: F1,67 = 0.042, P = 0.839)
and treatment groups (ANOVA: F2,67 = 0.0002, P = 0.999) produced
offspring of equal sex ratio. L/fast females produced offspring that
were significantly longer and heavier than M/slow offspring (Leastsquare means for SVL- L = 191.8 ± 1.74 mm, M = 181.5 ± 1.71 mm; leastsquare means for bodymass - L = 2.67 ± 0.08g, M = 2.44 ± 0.07g). Female
offspring from corticosterone-treated dams were significantly shorter
than all other groups of offspring (182.0± 1.5mm vs. 188.8 ± 1.6mm for
all other groups). (Table 3).
Locomotor performance
Analyses were based on relative speed, which is a measure of body
lengths per second. For sprint speed (speed over 1 m), males were
significantly faster than females (Table 3: least-square means for
males: 0.68 ± 0.02 lengths/s; females: 0.64 ± 0.01 lengths/s). As well,
individuals in two of three M/slow populations exhibited faster sprint
slither speeds than all of the L/fast populations. One L/fast population
Results
Corticosterone
Field levels
Samples were compared between the L/fast and M/slow pregnant
females to assess natural levels and variation of corticosterone. In
the field, pregnant females from M/slow sites had more than six
times higher levels of plasma corticosterone than pregnant females
from L/fast sites (least-square means for M/slow = 50.5 ± 7.9 ng/ml;
L/fast = 7.7 ± 15.3 ng/ml) (Table 2, Fig. 2).
Repeated measures
Analysis of the complete set of repeated measures of corticosterone for the four time points: arrival, days 1, 5 and 10 of the corticosterone manipulation revealed a significant rise in corticosterone
from arrival through day 1, and a plateau thereafter. This effect did not
differ between the two ecotypes, or with regard to corticosterone
application. (Table 2, Fig. 3).
Offspring levels
Corticosterone was assayed in pooled plasma from each resultant
litter. L/fast litters had significantly higher corticosterone levels than
M/slow litters. This pattern differed from that of their mothers where
M/slow dams had higher field levels than L/fast dams. Litters did not
differ in corticosterone with respect to dam treatment. (Table 3, Fig. 2).
Fig. 4. Percentage of all runs where (A) reversal behavior was performed, and (B) tail
lashing behavior was performed in offspring from mothers originating from either
Lakeshore [L/fast] or Meadow [M/slow] ecotypes. The different maternal treatments are
untreated (white bars) and CORT treated (black bars). NS — not significant, ⁎P b 0.05,
⁎⁎P b 0.001.
K.A. Robert et al. / Hormones and Behavior 55 (2009) 24–32
had significantly slower mean slither speed than any other population.
No differences were seen in burst slither (25 cm) speed.
Escape behaviors
Treatment significantly influenced escape behaviors, dependent
upon maternal ecotype (Fig. 4). Within the L/fast ecotype, offspring
of corticosterone-treated mothers displayed fewer reversals (Gstatistic = 5.35, P = 0.02) and similar tail lashing (G-statistic = 0.19,
P = 0.66) than offspring from untreated mothers. Within the M/slow
ecotype, offspring of corticosterone-treated mothers displayed similar
reversals (G-statistic = 2.03, P = 0.15), but decreased tail lashing (Gstatistic =12.01, P=0.0005) than offspring from untreated mothers. By
the time neonates are 3-month old, they attempt a reversal in 30% of
locomotor trials (K A Robert and A M Bronikowski, unpublished data).
That this frequency is three times higher than the frequencies observed
in this study of 1-month olds (ca. 5–10%) suggests an ontogenetic
trajectory of escape-type behaviors within the first year of life.
Discussion
Dam and offspring corticosterone levels
Dams from the meadow/slow-growth [“M/slow”] ecotype had
higher baseline plasma corticosterone than dams from the lakeshore/
fast-growth [“L/fast”] ecotype (Fig. 2). This pattern was consistent
across source population from within each ecotype. Previous work on
these ecotypes has shown that dry years impact food availability in
meadow, but not lakeshore, habitats (Bronikowski and Arnold, 1999).
M/slow snakes rely on anuran tadpoles and metamorphs as their food
source, which are only present in years with enough standing water
for anuran breeding (ca. 500 mm precipitation, Bronikowski and
Arnold, 1999). Interestingly, the likelihood of this critical precipitation
amount and, hence, of standing ponds with breeding anurans, is only
50% in any given year. Thus, in M/slow populations, corticosterone
may function in their marginal habitats to mobilize foraging behavior
in years when food is available. That glucocorticoids can function to
enhance an individual's survival through increased foraging or
metabolism has been documented in various birds (e.g., Kitaysky et
al., 2005; reviewed in Landys et al., 2006). Furthermore, once M/slow
females attain sexual maturation (ca. 400 mm SVL), they cease
growing and channel their energy into reproduction (Sparkman et al.,
2007). In contrast, L/fast snakes grow over their entire lifespan. Plasma
insulin-like growth factor 1 (IGF1) levels mirror these two reproductive strategies in L/fast and M/slow dams (Sparkman et al. in press).
Thus, meadow habitats with unpredictable food availability are
characterized by animals with: higher baseline glucocorticoids, higher
foraging behavior, and energy allocation directed at reproduction and
somatic maintenance (and not growth) (Bronikowski, in press).
Lakeshore habitats, in contrast, with predicable and high food
availability, are characterized by animals with: lower baseline
corticosterone, constant but lower foraging behavior, and energy
allocation directed at growth and reproduction. In 2006, the year that
these animals were collected, bountiful prey was available across all of
the populations of M/slow and L/fast snakes. Thus we would expect
this baseline difference in corticosterone between ecotypes (M/
slow N L/fast) to be even more pronounced in less favorable years. As
has been hypothesized by others, the role of corticosterone may shift
from mobilizing foraging to directing an all-out stress response (e.g.,
decreased immune function (Ricklefs, 2006), decreased reproduction
(Wingfield and Sapolsky, 2003)).
The trend that M/slow dams had higher baseline corticosterone
levels than L/fast dams was opposite that seen in the babies of
these mothers; L/fast babies had higher levels of corticosterone
than M/slow babies. A caveat to this comparison is that the dam
levels were obtained in the field, whereas the offspring values were
29
obtained in the laboratory. Perhaps a more relevant comparison
would be the pre-treatment levels for the dams upon arrival to the
laboratory. In this case, L/fast dams had slightly but significantly
higher values of corticosterone than M/slow dams. That captive
measures of corticosterone were higher in L/fast dams and neonates
suggests that the HPA/HPI axis may be more reactive in L/fast than
M/slow animals. Earlier common garden studies of neonates from
these two ecotypes showed that growth is faster over the first year
of life in L/fast babies under equivalent food-intake and temperature (Bronikowski, 2000). That corticosterone is present at higher
concentrations in neonates for whom fast growth is at a premium
provides additional support to the hypothesis that corticosterone
modulates the foraging behavior and/or energy mobilization of
these animals.
Neonatal reptiles are often born with yolk reserves or yolk plugs. In
our study system, newborn garter snakes have substantive yolk
reserves; in the wild, they do not feed until the spring following their
birth (A. M. Bronikowski, unpublished data). Whether this results
from the absence of appropriate prey or the absence of a feeding signal
from the brain is relevant to this study. For example, the hormone
leptin signals the brain to stop eating; it has been shown in at least one
reptilian study to have a seasonal pattern of low levels in the fall when
fat stores are expected to be high (Spanovich et al. 2006.) Leptin may
also be suppressed by glucocorticoids, which suggests that higher
baseline corticosterone levels may impede leptin signaling and
thereby mobilize foraging earlier in life. Although disentangling yolk
reserves, foraging propensity, and corticosterone levels in these fastand slow-growth ecotypes is beyond the scope of this study, a follow
up study measuring the simultaneous secretion of leptin and
corticosterone along with yolk reserve energetics is warranted.
Corticosterone manipulation: effects on dams
In this experiment, we failed to elevate plasma corticosterone
concentration of pregnant females that received exogenous corticosterone over a 10-day period. The effect of exogenous corticosterone
may have left a signature in the blood plasma concentrations that we
missed. Corticosterone application was in the evenings whereas blood
draws for corticosterone measurement were in the mornings. The
corticosterone circadian pattern in mammals (Krieger, 1973; Ottenweller et al., 1979) and birds (Tarlow et al., 2003) is to rise in the active
phase or just prior to the active phase and fall in the inactive phase. A
similar pattern has been observed in several lizard species (Chan and
Callard, 1972; Dauphin-Villemant and Xavier, 1987; Woodley et al.,
2003). If, for example, corticosterone levels rose and remained high
for treated females, sampling blood at the time of peak corticosterone
levels would suggest no effect yet the integrated levels would be
higher in treated animals.
Exogenous application, at these low concentrations, may simply
not impact free plasma corticosterone. This could occur if, for example,
levels of corticosterone binding globulin (CBG) are high and prevent
corticosterone from being free and biologically active (Slaunwhite
et al., 1962; Hammond, 1995). However, exogenous corticosterone
may temporarily elevate plasma levels above the binding capacity of
CBG resulting in free steroid, which could find its way to developing
embryos. An inability to regulate levels of glucocorticoid receptor (GR)
is another factor that may contribute to greater vulnerability of
embryos in females with small corticosterone increases. Intrauterine
growth impairment in rats due to maternal corticosterone may be due
to the embryos inability to down regulate GR when exposed to high
glucocorticoid levels (Ghosh et al., 2000).
Stressors associated with reproduction may be long term and
predictable, and may evoke an evolved response that differs from the
short term activation of the HPA/HPI in response to an unpredictable
stressor (e.g., predation attempt). This concept, that life-history
related stresses and unpredictable external stresses may require
30
K.A. Robert et al. / Hormones and Behavior 55 (2009) 24–32
differential modulation of the HPA/HPI axis, has been termed
allostasis (reviewed in Landys et al., 2006). Increases in plasma
corticosterone levels in pregnant T. elegans may be a normal part of
their pregnancy, however, we would require measurements from nonpregnant individuals over the same time period to determine if the
corticosterone increases are due to pregnancy itself. In viviparous
reptiles the increased energy demands of pregnancy and parturition
may require elevated corticosterone levels to help mobilize energy
stores and regulate the timing of parturition (Robert and Thompson,
2000; Meylan et al., 2003; reviewed in Moore and Jessop, 2003).
Alternatively, levels may have been elevated due to transfer to the
laboratory. In either case, additional application of corticosterone was
insufficient to raise plasma corticosterone further.
Notwithstanding, there was a clear effect of corticosterone
treatment on reproductive success. Corticosterone-treated mothers
had higher rates of stillborns and offspring that died within the first
month of life (29%) than did non-treated females (14%). Elevated
plasma corticosterone or hyperactivity of the maternal HPA/HPI axis in
mammals and reptiles elevates embryonic mortality (Davis and Plotz,
1954; Cree et al., 2003). Our results agree.
Corticosterone manipulation: maternal effects on offspring
Offspring from corticosterone-treated mothers were not sex biased
and performed equally well in locomotor trials. Female offspring from
corticosterone-treated dams were significantly shorter than all other
groups of offspring. This is particularly notable given that females tend
to quickly outgrow males in the laboratory, they are of larger body size
(SVL and mass) than males throughout their adult lifetimes, and they
have enhanced fitness at larger body sizes (Bronikowski and Arnold,
1999; Bronikowski, 2000; Sparkman et al., 2007). This is true within
both ecotypes, but the magnitude of the effect of SVL and body mass on
female fitness is greater for L/fast snakes. Thus, the reduction in size for
female offspring of corticosterone-treated mothers would have a greater
deleterious impact in lakeshore habitats than in meadow habitats.
Offspring from corticosterone manipulated mothers displayed
differences in escape behaviors. These altered behavioral phenotypes
were dependent upon maternal ecotype. Corticosterone-treated
mothers from L/fast populations produced offspring that exhibited
significantly less reversing. Corticosterone-treated mothers from M/
slow populations produced offspring with significantly more frequent
tail lashing. Reversal of locomotion direction and tail lashing are
categorized as anti-predation behaviors. The possible role of predatorescape behaviors is likely non-trivial based on the gamut of predators
that these snakes are subjected to on a daily basis during the active
season. We have observed that juvenile western terrestrial garter
snakes are prey items for a wide variety of vertebrate predators,
including avian (e.g., hawks, black birds, crows), mammalian (e.g.,
shrews, weasels), and reptilian (e.g., ophagous snakes) (A. M.
Bronikowski and S. J. Arnold, unpublished data). Although the
importance of these snakes in the diets of vertebrate predators is
unknown, the importance of predation on snakes from the perspective
of snake demography is quantifiable. We have reported higher annual
mortality rates for all life-history stages in L/fast populations compared
to M/slow populations (Bronikowski and Arnold, 1999; Sparkman et
al., 2007). Our current field research suggests that the primary cause of
the mortality differences between the two ecotypes is avian predation
(A. M. Bronikowski and S. J. Arnold, unpublished data).
In lakeshore populations, reversal of locomotion direction is a
prime behavioral tactic for capture-avoidance, a behavior that we
rarely document in meadow habitats. Thus, decreased reversal in
lakeshore sites is presumably detrimental based on the natural
repertoire of behaviors in this ecotype. Furthermore, our results
suggest that tail lashing, and its ecological context, is an additional
behavioral variable that warrants further study. For example, it may
differ between the two ecotypes, particularly in years when external
stressors (e.g., food shortage), and presumably dam corticosterone
concentrations, are greater in meadow sites. These same offspring
with either less reversal or less tail lashing behavior had sprint speeds
similar to those off offspring from untreated dams. A reduction in
escape behaviors may have helped offspring of corticosterone-treated
mothers remain as fast as control offspring in sprint performance.
When confronted by a predator, several strategies can be adopted: 1)
flee to shelter, 2) rely on crypsis, 3) fight back with aggression, or 4)
implement a diversion. In the case of fleeing, being fast may be more
important than in the other situations, given that these other
strategies do not depend on speed.
Brodie (1989, 1992, 1993) has shown that garter snakes (T. ordinoides) with checkered color patterns reverse direction more than
snakes with uniform pattern. In general, animals with disruptive color
patterns (e.g., checkered) are predicted to rely upon reversal and
crypsis more than animals with uniform color patterns, which are
predicted to rely on speed (Jackson et al., 1976; Pough, 1976; Creer,
2005). In our system L/fast snakes are checkered, and M/slow snakes
are uniformly black with a single yellow dorsal stripe. Based on this
color pattern difference and on the above-cited studies, the elimination of reversal behavior in L/fast offspring when their mothers have
elevated corticosterone would have fitness costs. One consequence of
lower plasma corticosterone in L/fast dams in nature (6-times lower in
this study) may be that offspring maintain their normal anti-predation
behavioral repertoire.
Recent studies of glucocorticoid levels and behavior in juvenile
Belding's ground squirrels suggest an intriguing possibility in these
snakes. Juvenile squirrels show 2-week surges of cortisol upon
emergence from their nests at the time that they are first learning
anti-predator and foraging behaviors (Mateo 2006). In addition, the
duration and level of cortisol levels in adult squirrels varies in a
predictable manner with predation risk across the landscape; lower
levels of cortisol are secreted in lower predation habitats (Mateo
2007). Mateo (2007) offers an interesting interpretation that habitat
quality and predation risk may mold glucocorticoid secretion,
specifically upregulating adrenal function at early developmental
and learning stages and during the adult stage. That neonate garter
snakes, exposed to altered circulating maternal corticosterone levels
exhibit altered innate anti-predator behaviors suggests that indirect
maternal effects may also be important in the shaping of the HPA/HPI
axis with respect to anti-predator behavior.
Corticosterone manipulation during pregnancy in reptiles has been
studied in common lizards, L. vivipara and in a viviparous gecko,
Hoplodactylus maculatus. These studies report a variety of effects of
corticosterone on offspring. For example, an effect on morphology and
performance (Meylan and Clobert, 2004, 2005; Uller et al., 2005;
Vercken et al., 2007) is common. As well, these researchers have found
that corticosterone treatment of mothers had a variety of effects on
offspring: (1) promoted philopatric behavior (De Fraipont et al., 2000;
Meylan et al., 2002); (2) promoted increased basking and activity (De
Fraipont et al., 2000; Belliure et al., 2004); and (3) promoted cautious
behavior, including retreat, and lowered activity following a simulated
attack (De Fraipont et al., 2000; Uller and Olsson, 2006). The timing of
exposure was not constant in these studies (Vercken et al., 2007), so
the array of offspring effects is suggestive of a suite of potential
maternal effects on offspring. The unknown influence of stage and
duration of exposure to corticosterone in squamate reptiles warrants
further investigation.
Acknowledgments
We thank Amanda Sparkman, Mathew Morrill, and Ann Cannon
who assisted with capture of females and collection of blood samples
at Eagle Lake. We are grateful to the state of California Dept. of Fish and
Game for scientific collecting permits. This project was supported by a
National Science Foundation Grant (DEB-0323379) to AMB.
K.A. Robert et al. / Hormones and Behavior 55 (2009) 24–32
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