The effects of maternal corticosterone levels on offspring behavior in fast- and slow-growth garter snakes (Thamnophis elegans)

Hormones and Behavior 55 (2009) 24–32

Contents lists available at ScienceDirect

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

References Aerts, P., Van Damme, R., Van Hooydonck, B., Zaaf, A., Herrel, A., 2000. Lizard locomotion: how morphology meets ecology. Neth. J. Zool. 50, 261–277. Arnold, S.J., 1983. Morphology, performance and fitness. Am. Zool. 23, 347–361. Arnold, S.J., Peterson, C.R., 2002. A model for reaction norms: the case of the pregnant garter snake and her temperature sensitive embryos. Am. Nat. 160 (3), 306–316. Barlow, S.M., Quyyumi, A.A., Rajaratnam, D.V., Sullivan, F.M., 1976. Effects of stress and adrenocorticotropin administration on plasma corticosterone levels at different stages of pregnancy in the mouse. Cell. Mol. life Sci. 32 (11), 1480–1481. Belliure, J., Meylan, S., Clobert, J., 2004. Prenatal and postnatal effects of corticosterone on behavior in juveniles of the common lizard, Lacerta vivipara. J. Exp. Zool. 301A, 401–410. Belthoff, J.R., Dufty Jr., A.M., 1995. Activity levels and the dispersal of western screechowls, Ornus kennicottii. Anim. Behav. 50, 558–561. Bennett, A.F., Huey, R.B., 1990. Studying the evolution of physiological performance. In: Futuyma, D.J., Antonovics, J. (Eds.), Oxford Surveys in Evolutionary Biology, Vol. 6. Oxford University Press, Oxford, U.K., pp. 251–284. Brodie III, E.D., 1989. Genetic correlations between morphology and antipredator behavior in natural populations of the garter snake Thamnophis ordinoides. Nature 342, 542–543. Brodie III, E.D., 1992. Correlational selection for color pattern and antipredator behavior in the garter snake Thamnophis ordinoides. Evolution 46, 1284–1298. Brodie III, E.D., 1993. Consistency of individual differences in anti-predator behavior and color pattern in the garter snake, Thamnophis ordinoides. Anim. Behav. 45, 851–861. Brodie III, E.D., Russell, N.H., 1999. The consistency of individual differences in behavior: temperature effects on antipredator behavior in garter snakes. Anim. Behav. 57, 445–451. Bronikowski, A.M. (in press) The evolution of aging phenotypes in snakes: a review and synthesis with new data. AGE J. Amer. Aging Society. doi10.1007/s11357-008-9060-5 Bronikowski, A.M., Arnold, S.J., 1999. The evolutionary ecology of life history variation in the garter snake Thamnophis elegans. Ecology 80 (7), 2314–2325. Bronikowski, A.M., 2000. Experimental evidence for the adaptive evolution of growth rate in the garter snake Thamnophis elegans. Evolution 54, 1760–1767. Bronikowski, A.M., Carter, P.A., Swallow, J.G., Girard, I.A., Rhodes, J.S., Garland Jr., T., 2001. Open-field behavior of house mice selectively bred for high voluntary wheel running. Behav. Genet. 31, 309–316. Chan, S.W., Callard, I.P., 1972. Circadian rhythm in the secretion of corticosterone by the desert iguana, Dipsosaurus dorsalis. Gen. Comp. Endocrinol. 18, 565–568. Christian, K.A., Tracy, R., 1981. The effect of the thermal environment on the ability of hatchling Galapagos land iguanas to avoid predation during dispersal. Oecologia 49, 218–223. Clark, M.M., Galef, B.G., 1998. Prenatal influences on reproductive behavior of adult rodents. In: Mousseau, T.A., Fox, C.W. (Eds.), Maternal Effects as Adaptations. Oxford University Press, pp. 261–271. Cree, A., Tyrrell, C.L., Preest, M.R., Thornburn, D., Guillette, L.J., 2003. Protecting embryos from stress: corticosterone effects and the corticosterone response to capture and confinement during pregnancy in a live-bearing lizard (Hoplodactylus maculates). Gen. Comp. Endocrinol. 134, 316–329. Creer, D.A., 2005. Correlations between ontogenetic change in color pattern and antipredator behavior in the racer, Coluber constrictor. Ethology 111, 287–300. Dauphin-Villemant, C., Xavier, F., 1987. Nychthemeral variations of plasma corticosteroids in captive female Lacera vivipara Jacquin: influence of stress and reproductive state. Gen. Comp. Endocrinol. 67, 292–302. Davis, M.E., Plotz, E.J., 1954. The effects of cortisone acetate on intact and adrenalectomized rats during pregnancy. Endocrinologica 54, 384–395. De Fraipont, M., Clobert, J., John-Alder, H., Meylan, S., 2000. Increased pre-natal maternal corticosterone promotes philopatry of offspring in common lizards Lacerta vivipara. J. Anim. Ecol. 69, 404–413. Downes, S.J., Shine, R., 1999. Do incubation-induced changes in a lizard's phenotype influence its vulnerability to predators? Oecologia 120 (1), 9–18. Dufty, A.M., Clobert, J., Møller, A.P., 2002. Hormones, developmental plasticity and adaption. Trends. Ecol. Evol. 17, 190–196. Ford, N., 1996. Behaviour of garter snakes. In: Rossman, D.A., Ford, N.B., Seigel, R.A. (Eds.), The Garter Snakes: Evolution and Ecology. The University of Oklahoma Press, pp. 90–116. Garland, T.J., 1999. Laboratory endurance capacity predicts variation in field locomotor behaviour among lizard species. Anim. Behav. 58, 77–83. Garland Jr., T., Freeman, P.A., 2005. Selective breeding for high endurance running increases hindlimb symmetry. Evolution 59, 1851–1854. Garland Jr., T., Losos, J.B., 1994. Ecological morphology of locomotor performance in squamate reptiles. In: Wainwright, P.C., Reilly, S.M. (Eds.), Ecological Morphology: Integrative Organismal Biology. University of Chicago Press, Chicago, pp. 240–302. Ghosh, B., Wood, C.R., Held, G.A., Abbott, B.D., Lau, C., 2000. Glucocorticoid receptor regulation in the rat embryo: a potential site for developmental toxicity. Toxicol. Appl. Pharmacol. 156, 221–229. Greenberg, N., Wingfield, J.C., 1987. Stress and reproduction: reciprocal relationships. In: Norris, D.O., Jones, R.E. (Eds.), Hormones and Reproduction in Finches, Amphibians and Reptiles. Plenum Press, New York. Guillette Jr., L.J., Cree, A., Rooney, A.A., 1995. Biology of stress: interactions with reproductions, immunology and intermediary metabolism. In: Warwick, C., Frye, F.L., Murphy, J.B. (Eds.), Health and Welfare of Captive Reptiles. Chapman and Hall, London, pp. 32–81. Hammond, G.L., 1995. Potential functions of plasma steroid-binding proteins. Trends Endrocrinol. Metab. 6 (9/10), 298–304.

31

Huey, R.B., Peterson, C.R., Arnold, S.J., Porter, W.P., 1989. Hot rocks and not-so-hot rocks: retreat site selection by garter snakes and its thermal consequences. Ecology 70 (4), 931–944. Husak, J.F., Fox, S.F., Loven, M.B., Van Den Bussche, R.A., 2006. Faster lizards sire more offspring: sexual selection on whole-animal performance. Evolution 60 (10), 2122–2130. Irschick, D.J., Garland Jr., T., 2001. Integrating function and ecology in studies of adaption: investigations of locomotor capacity as a model system. Annu. Rev. Ecol. Syst. 32, 367–396. Jackson, J.F., Ingram III, W., Campbell, H.W., 1976. The dorsal pigmentation pattern of snakes as an antipredator strategy: a multivariate approach. Am. Nat. 110, 1020–1053. Janzen, F., 1995. Experimental evidence for the evolutionary significance of temperature-dependent sex determination. Evolution 49 (5), 864–873. Jayne, B.C., Bennett, A.F., 1990. Selection on locomotor performance capacity in a natural population of Garter Snakes. Evolution 44 (5), 1204–1229. Kephart, D.G., Arnold, S.J., 1982. Garter snake diets in a fluctuating environment: a seven-year study. Ecology 63, 1232–1236. Kitaysky, A.S., Romano, M.D., Piatt, J.F., Wingfield, J.C., Kikuchi, M., 2005. The adrenocortical response of tufted puffin chicks to nutritional deficits. Horm. Behav. 47, 606–619. Knapp, R., Moore, M.C., 1997. A non-invasive method for sustained elevation of steroid hormone levels in reptiles. Herpetol. Rev. 28, 33–36. Krieger, D.T., 1973. Effect of ocular enucleation and altered lighting regimens at various ages on the circadian periodicity of plasma corticosteroid levels in the rat. Endocrinology 93, 1077–1091. Lance, V.A., 1990. Stress in reptiles. In: Epple, A., Scanes, C.G., Stetson, M.H. (Eds.), Prospects in Comparative Endocrinology. Wiley-Liss, New York, pp. 461–466. Landys, M.M., Ramenofsky, M., Wingfield, J.C., 2006. Actions of glucocorticoids at a seasonal baseline as compared to stress-related levels in the regulation of periodic life processes. Gen.Comp. Endocrinol. 148, 132–149. Manier, M.K., Arnold, S.J., 2006. Ecological correlates of population genetic structure: a comparative approach using a vertebrate metacommunity. Proc. R. Soc. B. 273 (1604), 3001–3009. Manier, M.K., Seylor, C.M., Arnold, S.J., 2007. Adaptive divergence between ecotypes of the terrestrial garter snake, Thamnophis elegans, assessed with Fst–Qst comparisons. J. Evol. Biol. 20 (5), 1705–1719. Mason, R.T., 1992. Reptilia pheromones. In: Gans, C., Crews, D. (Eds.), Biology of the Reptilian, vol. 18. Chicago Univ. Press, Chicago, pp. 114–228. Mateo, J.M., 2006. Developmental and geographic variation in stress hormones in wild Belding's ground squirrels (Spermophilus beldingi). Horm. Behav. 50, 718–725. Mateo, J.M., 2007. Ecological and hormonal correlates of antipredator behavior in adult Belding's ground squirrels (Spermophilus beldingi). Behav. Ecol. Sociobiol. 62, 37–49. McEwen, B.S., Wingfield, J.C., 2003. The concept of allostasis in biology and biomedicine. Horm. Behav. 43, 2–15. Meylan, S., Clobert, J., 2004. Maternal effects on offspring locomotion: influence of density and corticosterone elevation in the lizard Lacerta vivipara. Physiol. Biochem. Zool. 77, 450–458. Meylan, S., Clobert, J., 2005. Is corticosterone mediated phenotype development adaptive? Horm. Behav. 48, 44–52. Meylan, S., Belliure, J., Clobert, J., de Fraipont, M., 2002. Stress and body condition as prenatal and postnatal determinants of dispersal in the common lizard (Lacerta vivipara). Horm. Behav. 42, 319–326. Meylan, S., Dufty, A.M., Clobert, J., 2003. The effect of transdermal corticosterone application on plasma corticosterone levels in pregnant Lacerta vivipara. Comp. Biochem. Physiol. A. 134, 497–503. Miles, D.B., 2004. The race goes to the swift: fitness consequences of variation in sprint performance in juvenile lizards. Evol. Ecol. Res. 6, 63–75. Miles, D.B., Calsbeek, R., Sinervo, B., 2007. Corticosterone, locomotor performance and metabolism in side blotched lizards (Uta stansburiana). Horm. Behav. 51 (4), 548–554. Moore, I.T., Jessop, T.S., 2003. Stress, reproduction, and adrenocortical modulation in amphibians and reptiles. Horm. Behav. 43, 39–47. Mousseau, T.A., Fox, C.W., 1998. Maternal Effects as Adaptations. Oxford University Press. Neter, J., Wasserman, W., Kutner, M.H., 1990. Applied Linear Statistical Models, 3rd ed. Irwin Press, Boston. Neumann, I.D., Johnstone, H.A., Hatzinger, M., Liebsch, G., Shipston, M., Russell, J.A., Landgraf, R., Douglas, A.J., 1998. Attenuated neurendocrine responses to emotional and physical stressors in pregnant rats involve adenohypophysial changes. J. Physiol. 508, 289–300. Norris, D.O., Jones, R.E., 1987. Hormones and Reproduction in Fishes, Amphibians and Reptiles. Plenum Press, New York. Ottenweller, J.E., Meier, A.H., Russo, A.C., Frenke, M.E., 1979. Circadian rhythms of plasma corticosterone binding activity in the rat and the mouse. Acta Endocrinol. 91, 150–157. Painter, D., Jennings, D.H., Moore, M.C., 2002. Placental buffering of maternal steroid hormone effects on fetal and yolk hormone levels: a comparative study of a viviparous lizard, Sceloporus jarrovi, and an oviparous lizard, Sceloporus graciosus. Gen. Comp. Endocrinol. 127, 105–116. Pough, F.H., 1976. Multiple cryptic effects of crossbanded and ringed patterns of snakes. Copeia 1976, 834–836. Rezende, E.L., Kelly, S.A., Gomes, F.R., Chappell, M.A., Garland Jr, T., 2006. Effects of size, sex, and voluntary running speeds on costs of locomotion in lines of laboratory mice selectively bred for high wheel running activity. Physiol. Biochem. Zool. 79, 83–99.

32

K.A. Robert et al. / Hormones and Behavior 55 (2009) 24–32

Reznick, D.N., Bryant, M.J., Roff, D., Ghalambar, C.K., Ghalambar, D.E., 2004. Effect of extrinsic mortality on the evolution of senescence in guppies. Nature 431, 1048. Ricklefs, R.E., 2006. Embryo development and ageing in birds and mammals. Proc. Royal Soc. London B. 273, 2077–2082. Robert, K.A., Thompson, M.B., 2000. Energy consumption by embryos of a viviparous lizard, Eulamprus tympanum, during development. Comp. Biochem. Physiol. A. 127, 481–486. Robert, K.A., Brunet-Rossinni, A., Bronikowski, A.M., 2007. Testing the “free radical theory of aging” hypothesis: physiological differences in long lived and short lived Colubrid snakes. Aging Cell. 6, 395–404. Romero, L.M., 2002. Seasonal changes in plasma glucocorticoid concentrations in freeliving vertebrates. Gen. Comp. Endocrinol. 128, 1–24. Slaunwhite, W.R., Lockie, G.N., Black, N., Sandberg, A.A., 1962. Inactivity in vivo of transcortin-bound cortisol. Science 135, 1062–1063. Sokal, Rohlf, 1981. Biometry, 2nd ed. Freeman, New York. Spanovich, S., Niewiarowski, P.H., Londraville, R.L., 2006. Seasonal effects on circulating leptin in the lizard Sceloporus undulatus from two populations. Comp. Biochem. Physiol. B: Biochem. Molec. Biol. 143, 507–513. Sparkman, A.M., Arnold, S.J., Bronikowski, A.M., 2007. An empirical test of evolutionary theories for reproductive senescence and reproductive effort in the garter snake Thamnophis elegans. Proc. R. Soc. B. 274 (1612), 943–950. Sparkman, A.M., Vleck, C., and Bronikowski, A.M. (in press) The evolutionary ecology of endocrine-mediated life history variation in the garter snake. Thamnophis elegans. Ecology 89.

Tarlow, E.M., Hau, M., Anderson, D.J., Wikelski, M., 2003. Diel changes in plasma melatonin and corticosterone concentrations in tropical Naza boobies (Sula granti) in relation to moon phase and age. Gen. Comp. Endocrin. 133 (3), 297–304. Uller, T., Meylan, S., de Fraipont, M., Colbert, J., 2005. Is sexual dimorphism affected by the combined action of prenatal stress and sex ratio? J. Exp. Zool. 303, 1110–1114. Uller, T., Olsson, M., 2006. Direct exposure to corticosterone during embryonic development influences behavior in an ovoviviparous lizard. Ethology 112, 390–397. Vercken, E., de Fraipont, M., Dufty, A.M., Clobert, J., 2007. Mother's timing and duration of corticosterone exposure modulate offspring size and natal dispersal in the common lizard (Lacerta vivipara). Horm. Behav. 51, 379–386. Warner, D.A., Andrews, R.M., 2002. Laboratory and field experiments identify sources of variation in phenotypes and survival of hatchling lizards. Biol. J. Linn. Soc. 76, 105–124. Wingfield, J.C., Vleck, C.M., Moore, M.C., 1992. Seasonal changes of the adrenocortical response to stress in birds of the Sonoran Desert. J. Exp. Zool. 264, 419–428. Wingfield, J.C., Sapolsky, R.M., 2003. Reproduction and resistance to stress: When and how. J. Neuroendocrinol. 15, 711–724. Woodley, S.K., Moore, M.C., 2002. Plasma corticosterone response to an acute stressor varies according to reproductive condition in female tree lizards (Urosaurus ornatus). Gen. Comp. Endocrinol. 128, 143–148. Woodley, S.K., Painter, D.L., Moore, M.C., Wikelski, M., Romero, L.M., 2003. Effect of tidal cycle and food intake on the baseline plasma corticosterone rhythm in intertidally foraging marine iguanas. Gen. Comp. Endocrinol. 132, 216–222.