Fecal glucocorticoid metabolites of experimentally stressed captive and free-living starlings: Implications for conservation research

General and Comparative Endocrinology 158 (2008) 20–28

Contents lists available at ScienceDirect

General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen

Fecal glucocorticoid metabolites of experimentally stressed captive and free-living starlings: Implications for conservation research Nicole E. Cyr *, L. Michael Romero Department of Biology, Tufts University, Medford, MA 02155, USA

a r t i c l e

i n f o

Article history: Received 11 September 2007 Revised 24 February 2008 Accepted 2 May 2008 Available online 10 May 2008 Keywords: Fecal glucocorticoid metabolites Chronic stress Conservation biology European starlings

a b s t r a c t Fecal glucocorticoid metabolite (FGM) analysis has received considerable attention in conservation biology because it has potential to be used as a noninvasive measure of stress in animals. There has been a recent and extensive literature describing the importance of technical, physiological, and biological validations of this technique, yet surprisingly little is known about how FGM concentrations change during chronic stress. Therefore, we experimentally induced chronic stress in both captive and free-living European starlings (Sturnus vulgaris). Chronic stress was elicited using a rotation of four different 30 min acute stressors for 16 days in the laboratory and 8 days in the field. Exogenous ACTH, the primary glucocorticoid secretagog, significantly increased FGM concentrations in approximately 2 h, and our assay detected endogenous diel glucocorticoid rhythms similar to those of other birds. Thus, our assay was both physiologically and biologically validated. However, experimentally induced chronic stress did not alter daytime or nighttime FGM concentrations in captive starlings. In contrast, chronically stressed adult female starlings had higher FGM concentrations than unstressed female starlings in the field. Our field data support the general assumption that higher FGM concentrations indicate chronic stress, but our captive data do not. Overall, our results suggest that more research is need before FGM analysis can be used as a reliable measure of stress in animals, especially those kept in captivity. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction All animals must cope with unpredictable environmental stressors. The stress response protects animals from unpredictable stressful events in the short-term (Sapolsky et al., 2000). However, long-term activation of the stress response to chronic stressors has many negative physiological consequences. During stress the hypothalamo-pituitary-adrenal (HPA) axis is activated to increase the synthesis and release of glucocorticoids (GCs) from the adrenal gland. Increased circulating GC concentrations mobilize stored glucose and alter behavior to facilitate survival during acute stress, but chronically elevated GCs are known to cause several harmful physiological conditions such as hyperglycemia, neuronal cell death, and suppression of the immune and reproductive systems (Sapolsky, 1992; Wingfield and Romero, 2001). Consequently, GC concentrations are commonly used to measure stress in animals. Since stress, as measured by GC release, is related directly to health, conservation biologists are interested in using GC concentrations to assess the health of individuals in a particular population to estimate the overall health of the population (Mostl and Palme, 2002). Indeed, several studies have used GCs as indicators of population health (Creel et al., 2002; Homan et al., 2003; * Corresponding author. Address: Chemistry Department, Wellesley College, 106 Central Street, Wellesley, MA 02481, USA. Fax: +1 617 627 3805. E-mail address: [email protected] (N.E. Cyr). 0016-6480/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2008.05.001

Hopkins et al., 1997; Wasser et al., 1997). Most studies have demonstrated an increase in GC concentrations with presumed stress (e.g., Arlettaz et al., 2007; Wasser et al., 1997), but others have reported the opposite (e.g., Homan et al., 2003). One factor that could explain the discrepancy in the results of these studies is whether GC concentrations were measured in blood or fecal samples. The majority of studies using GC concentrations as an indicator of population health have measured GCs in blood plasma. Although there are advantages to quantifying GCs in plasma, such as the ability to understand changes in adrenal responsiveness by using a capture and restraint protocol (Wingfield, 1994), there are also disadvantages associated with measuring GCs in blood. For example, blood samples represent a point sample that reveals the short-term hormone level, which may not represent the animal’s long-term physiological state. Furthermore, animals must be captured and handled to acquire the blood sample, which could affect the GC response. As a result, many researchers have chosen to measure GC metabolites excreted in fecal samples in lieu of blood (e.g., Creel et al., 1997a; Foley et al., 2001; Wasser et al., 1997). Fecal glucocorticoid metabolite (FGM) analysis is particularly appealing to conservation biologists because samples can be collected noninvasively, which is especially important when studying endangered species. For example, FGM analysis has been used to implicate physiological stress in a number of endangered species such as Northern spotted owls (Strix occidentalis caurina) (Wasser et al., 1997), African elephants

N.E. Cyr, L.M. Romero / General and Comparative Endocrinology 158 (2008) 20–28

(Loxodonta africana) (Foley et al., 2001), and African wild dogs (Lycaon pictus) (Creel et al., 1997a,b). However, there are several factors which confound the interpretation of FGM analysis (reviewed in Millspaugh and Washburn, 2004). Such problems include technical issues such as sample storage time, treatment and assay methods, ambient conditions such as temperature (Goymann et al., 2006), and biological issues such as well as the animal’s diet, age, body condition (Millspaugh and Washburn, 2004). It is also important to note that GCs are highly metabolized in the liver (Brownie, 1992). Therefore, when measuring GC concentrations in fecal samples one is actually measuring metabolites and not the intact steroid; hence, the term fecal glucocorticoid metabolites (Mostl et al., 2005). Several recent reviews have outlined methods for validating the use of FGM analysis (Goymann, 2005; Klasing, 2005; Mostl et al., 2005; Palme, 2005; Touma and Palme, 2005) and demonstrated that, once appropriately validated, FGM analyses can be used to accurately assess stress (Baltic et al., 2005; Hirschenhauser et al., 2005; Thiel et al., 2005; Wasser and Hunt, 2005). Another explanation for the discrepancy in the results of previous studies using GCs to estimate population health is that these studies have been largely correlative. For example, Wasser et al. (1997) found a correlation between proximity to logging roads and increased FGM concentrations in male Northern spotted owls. In contrast, Homan et al. (2003) found a negative correlation between presumed habitat quality and plasma GC concentrations in male spotted salamanders (Ambystoma maculatum). There have been few empirical studies that provide a direct causal link between poor health (i.e., stress) and altered GC concentrations in free-living animals. Typically, there is an a priori assumption that an environmental factor (such as proximity to logging roads) is stressful, and when GC concentrations positively correlate with that environmental factor, the causal link is assumed to exist. Rarely is the a priori assumption tested empirically (although see Arlettaz et al., 2007). One study; however, that experimentally induced chronic stress in free-living European starlings (Sturnus vulgaris) found that chronically stressed starlings had lower plasma GC concentrations and fledged fewer young (Cyr and Romero, 2007). This study demonstrated that chronic stress has fitness consequences in free-living animals, but that plasma GC concentrations may be a poor measure of chronic stress. The purpose of the present study was to extend Cyr and Romero (2007) by measuring FGM concentrations of unstressed and chronically stressed captive and free-living European starlings. Our goals were (1) to validate the use of FGM analysis for use in European starlings; (2) to measure FGM’s in unstressed starlings as well as starlings under acute and chronic stress conditions in the laboratory; and (3) to compare FGM’s of unstressed and chronically stressed starlings in the field. 2. Materials and methods 2.1. Study species European starlings were used for both laboratory and field studies. Starlings are a model species for this type of research because they are very common throughout the US, adapt well to captivity, and readily use nest boxes. Furthermore, starlings have commonly been used in studies of stress; thus, there is an extensive literature describing their stress response (e.g., Nephew and Romero, 2001, 2003; RemageHealey and Romero, 2001; Rich and Romero, 2005). All experiments were conducted according to AALAC guidelines and approved by the Institutional Animal Care and Use Committee at Tufts University. 2.2. Capture and housing for laboratory studies Twenty-four wild, nonbreeding, European starlings were caught using mist nets in eastern Massachusetts during the winter and immediately transported to a flight aviary at Tufts University (Medford, MA) where they were held on a short day 10L:14D light cycle to mimic winter conditions. Birds were later moved to an indoor experimental room and placed in individual cages (45  37  33 cm). The experi-

21

mental room was kept at 25 °C and the cages were arranged so that the birds were able to see and hear each other. Birds were a mixture of males and females and sex was ignored because previous work indicates that male and female captive starlings have equivalent stress responses (Romero and Remage-Healey, 2000). All birds were provided with water and food at all times for ad libitum consumption. 2.3. Sample collection and treatment For all laboratory experiments, we used absorbent paper to line the bottom of the cages in order to collect fecal samples and absorb as much of the urine portion of the sample as possible. The absorbent paper was changed twice a day (see below) and each time all the recoverable fecal pellets were combined to make a single sample for that collection period. In the field, fecal samples were collected opportunistically during capture and handling. For both field and laboratory studies the urate portion, which also contains GC metabolites (Mostl et al., 2005) was removed and the fecal portion was collected and immediately stored at 20 °C. Samples were homogenized, lyophilized to remove water without heating, and weighed to 0.1 g prior to the extraction. Millspaugh and Washburn (2004) and Goymann (2005) have shown that small fecal samples often produce disproportionately higher FGM concentrations than larger samples. We found that this was also the case for starlings. We pooled samples from 8 individuals and found that aliquots of the dried fecal pool weighing 0.5, 0.2, and 0.1 g resulted in similar FGM concentrations; however, aliquots weighing less than 0.1 g resulted in substantially higher FGM concentrations. Consequently, fecal samples smaller than 0.1 g were excluded from all studies. To extract the FGMs, we added 1 ml of 60% methanol to the lyophilized 0.1 g sample. The sample was then vortexed for 30 min, and centrifuged at 1000g for 10 min. The supernatant was removed to be assayed. This extraction technique has proven highly effective in other bird species (Touma and Palme, 2005). 2.4. FGM assay We used the 125I RIA kit from MP Biomedicals (cat. #07-120102; Costa Mesa, CA), formerly ICN Biomedicals. This assay kit is designed to measure plasma corticosterone, but has proven effective at measuring FGM concentrations in several species, including birds (Wasser et al., 2000). The details of this assay are described in Wasser et al. (2000). Briefly, we first created a standard curve of known unlabeled corticosterone concentrations which included (1000; 500; 250; 100; 50; 25; 12.5 ng/ml). For our sample tubes, we used 50 ll of sample supernatant. To both the standard curve and the sample tubes we and added 125I labeled corticosterone and corticosterone antibody. After a 2-h incubation period, precipitant was added to samples and the standard curve and all tubes were centrifuged at 1000g for 10 min. The supernatant was removed and the 125I remaining in the pellet was measured with a gamma counter. 2.5. Validation studies Eight starlings were used for technical validation of the FGM assay and physiological validation of FGM analysis. We used a 0.5-g fecal sample pooled from the eight birds to test the assay for parallelism and accuracy (Mostl et al., 2005). For the accuracy test we added 50 ll of sample supernatant to each standard provided by the kit to determine whether the addition of our supernatant significantly altered the percent binding curve. To test for parallelism, we created a twofold serial dilution of 100 ll of sample supernatant to compare the slope of our serially diluted sample to the slope of the standard curve provided by the assay kit. One of the most important validations studies for FGM analysis is the adrenocorticotropin hormone (ACTH) challenge (Touma and Palme, 2005). ACTH is the pituitary hormone that stimulates GC release from the adrenal cortex, and the ACTH challenge involves injecting a pharmacological dose of ACTH into the animal in order to test whether the subsequent increase in GCs can be detected by FGM analysis. ACTH has been shown to increase FGM concentrations in a number of mammals and birds (reviewed in Touma and Palme, 2005). For our ACTH challenge, we split 8 starlings into two groups of 4. Each group was given a 10 ll intramuscular (pectoral muscle) injection of ACTH (100 IU/kg body weight, dissolved in saline) as well as a saline control. Injections were administered using a counter-balanced design such that group 1 was injected with ACTH and group 2 injected with saline on day 1 and one week later the birds were given the opposite injection. Fecal samples were collected immediately prior to injection and every hour following injection for 4 h to determine the time lag between ACTH injection and increased GC metabolites in feces. The 100 IU/kg ACTH dose significantly increases plasma GC concentrations in starlings (Rich and Romero, 2005). Since the liver metabolizes GCs, it is important to determine that measured metabolites are of GCs and not other steroid hormones (Mostl et al., 2005). To test this, we injected a bolus (5 mg/kg body weight) of progesterone dissolved in peanut oil, as well as a peanut oil control using a counter-balance design, into the 8 birds and collected fecal samples 4 h post-injection. We chose progesterone because this is the steroid hormone most likely to be present in significant concentrations at the time of the experiment given that the birds were kept on short days (i.e., winter conditions) and estrogen and testosterone concentrations should be low.

22

N.E. Cyr, L.M. Romero / General and Comparative Endocrinology 158 (2008) 20–28

2.6. Chronic stress—captive study Fecal samples were collected at 2 time points daily before, during, and after chronic stress. These samples represented daytime FGMs (collected immediately before lights off at 17:00) and nighttime FGMs (collected at 08:00, lights on at 07:00) of birds that were unstressed, acutely stressed, and chronically stressed. For the initial collections the birds were undisturbed, except for routine husbandry and changing the collection papers, for 7 days (i.e., unstressed). Birds were then restrained in an opaque bag for 30 min to induce acute stress and collection continued for 24 h (i.e., 1 daytime and 1 nighttime collection). The birds were then subjected to a chronic stress protocol for 16 days with a daytime and nighttime sample collected each day. Finally the birds were left undisturbed for 5 days of recovery from the chronic stress with daytime and nighttime samples collected each day. Details of the chronic stress protocol have been published previously (Cyr et al., 2007; Rich and Romero, 2005). Briefly, we induced chronic stress by applying four acute 30 min stressors (loud radio, rocking the bird’s cage, tapping on the bird’s cage, introduction to a human voice, and restraint) randomly each day for 16 days. Stressors were spaced 1–1.5 h apart with the first stressor of the day administered more than 1–2 h after lights on and the last stressor of the day administered 1–2 h before lights off. Each stressor has been shown to increase circulating plasma GC concentrations to variable amounts (Nephew and Romero, 2003; Rich and Romero, 2005). Blood samples for baseline (immediately prior to the noon-time stressor) and stress-induced (immediately following the noontime stressor) plasma CORT analysis were taken from birds before and during the chronic stress protocol. In order to compare the changes that occur in plasma GC concentration to those that occur in FGM concentrations during chronic stress, we integrated the baseline and stress-induced changes in plasma GC concentrations that have been observed in starlings during the chronic stress protocol. Our previous studies have shown that starlings progressively decrease both baseline and stress-induced plasma GC concentrations during the chronic stress protocol (Nephew and Romero, 2003; Rich and Romero, 2005); however, we wanted to integrate the changes in plasma GCs over the course of the 10 h day to make the plasma GC concentrations more comparable to the FGM concentrations. To that end, we compiled data from each of these previous chronic stress trials (from this study, unpublished data, and data taken from Cyr et al., 2007), during which baseline plasma GC concentrations as well as restraint-induced plasma GC concentrations were measured. Sample sizes ranged form 7 to 19 birds for each day of the chronic stress period, Sample sizes varied because blood samples were not taken from all birds on all days. This analysis is an estimate of integrated plasma GC concentrations and may not reflect the true amount of GC secretion. We estimated the total amount of circulating GCs over the course of a day for unstressed, acutely stressed, and chronically stressed birds. First, for unstressed birds we multiplied the mean baseline GC concentrations of birds unstressed prior to bleeding by 10 (for 10 h of day). Second, for acutely stressed birds we determined the restraint-induced increase in GC concentrations (for a 30 min restraint, GC concentrations are elevated at 30 min and have returned to baseline 30 min after the end of restraint; Rich and Romero, 2005) and added that to the baseline concentrations. Third, we used our previous studies to determine the change in baseline and restraint-induced GC concentrations each day during the chronic stress protocol. For the chronically stressed birds in the present experiment, we knew which stressors the birds were exposed to each day and the GC response that each stressor should elicit (Rich and Romero, 2005; Romero and Remage-Healey, 2000). Since previous data indicate that GC responses to all stressors had ended 30 min after the end of the stressor (Rich and Romero, 2005), we assumed that the total hourly amount of GCs secreted was approximately ½ of the GC concentration at the end of the 30 min stressor. We then summed the responses for all four daily stressors, added this to the baseline GC concentration for that day (multiplied by 10 for the 10 h of the day) to get the estimated total amount of GCs secreted that day. Finally, we adjusted the four daily stress responses by the percent change in the GC response to restraint the occurred progressively over the 16 days of chronic stress (Cyr et al., 2007; Rich and Romero, 2005).

Average daytime plasma GCs ¼ ðBaseline GCs  10Þ þ ½ðGCðstressor 2Þ  0:5Þ  X þ ½ðGCðstressor 3Þ  0:5Þ  X ½ðGCðstressor 4Þ  0:5Þ  X;

ð1Þ

where X is the GC response to restraint on that particular day of chronic stress (see restraint-induced GC from Fig. 3) divided by the average response to restraint taken from nonchronically stressed starlings (see restraint-induced GC from Fig. 3). This approach provides a more integrated measure of plasma GCs than simple point samples and better reflects what can be metabolized by the liver and excreted in the feces, but includes several assumptions. The assumptions are: (1) that the baseline average does not change appreciably over the course of the day; (2) that the GC response to each individual stressor does not change over the course of the day (i.e., the GC response to the radio is the same in the morning as it is in the afternoon); (3) the change in GC response to restraint each day during chronic stress (i.e., X) is comparable to the change that occurs to each of the other acute stressors. Despite these assumptions, we believe that this integrated analysis is a better measure to compare with FGM results. It was necessary to estimate daily plasma GC concentra-

tions because it would have been impossible to take the necessary amount of blood to measure GCs throughout the course of a 10-h day, much less repeating this for each of the 16 days of chronic stress, in an 80 g starling. 2.7. Chronic stress—field study We studied a nest box population of starlings in Grafton, MA for two breeding seasons (2005 and 2006). We checked nests every other day until the first egg appeared and then every day until clutch completion. Nesting pairs were assigned to a control or experimental group in alternating chronological order. For the experimental group, we used Cyr and Romero’s (2007) protocol to induce chronic stress. Briefly, we rotated four (30 min) stressors spaced 1–2 h apart each day for 8 days. These stressors were a loud radio, predator calls, a novel object placed on the nest box, and four different predator decoys (snake, owl, falcon, or rat) positioned on or near the nest box. The chronic stress protocol was initiated after clutch completion in order to avoid nest abandonment given that the nesting pair had made an investment upon clutch completion. The control group was left undisturbed after determining clutch completion. We chose 8 days to reduce the chance of abandonment, but to ensure that the protocol elicited chronic stress. GC changes caused by chronic stress in captive starlings were evident 8 days after the onset of the chronic stress protocol, and persisted for 9 days following the completion of the chronic stress protocol (Rich and Romero, 2005). For this study, we focused on the females because they provide most of the parental care in this species and were thus less likely to abandon the nest during the chronic stress period (Cabe, 1993). Experimental females were subjected to 8 days of chronic stress and captured at the termination, 9 days after the onset of chronic stress. Control and experimentally stressed females were captured at the same time during the nesting cycle to control for time of capture. Adult females were captured at the nest box either incubating eggs or brooding young. Once captured, a fecal sample was taken and then females were banded with a U.S. Fish and Wildlife Service aluminum band and 3 color bands. Measurements of mass (to the nearest g), and tarsus length (to the nearest mm) were also taken. In addition to adult females, nestlings were also sampled. A fecal sample was taken from nestlings 16 days post hatch and nestlings were also banded with a U.S. Fish and Wildlife Service aluminum band. Measurements taken from nestlings included mass (to the nearest g), and tarsus length (to the nearest mm). All fecal samples were immediately placed on ice, and then transported to the Tufts Medford (MA) campus where they were stored at 20 °C. 2.8. Statistics We used JMP (version 5.0, SAS institute) and SAS (version 9.1) for statistical analyses. All data were tested for normal distribution using Shapiro-Wilk W goodness of fit test and homogenous variances using Levene’s test. For all variables, data were distributed normally (all p > 0.05) and variances were equal (all p > 0.05) except for the FGM data during chronic stress in the laboratory. For the laboratory chronic stress study we used a repeated measures design by comparing FGM changes in the same individuals prior to the chronic stress protocol. Consequently, SAS was used to first rank transform the data and then perform repeated measures ANOVA analyses for both daytime and nighttime laboratory FGM data. We also tested for sex differences in the data and found no sex difference for any variable (all p > 0.05), thus males ad females were pooled for all analyses. To validate the assay we tested for accuracy and parallelism. We used a general linear model to statistically determine whether these tests altered the slope of the standard curve. A two-way repeated measures ANOVA was used to analyze the results of the ACTH challenge, and Fisher’s PLSD was used for post hoc analyses. A paired t-test was used to analyze the progesterone data. Finally, a two tailed t-test was used to compare FGM concentrations of control and experimentally stressed adult females as well as to compare FGM concentrations of nestlings from experimentally stressed and unstressed broods in the field. All means are presented as ± their standard errors.

3. Results 3.1. Validation studies For the accuracy test we added 100 ll of sample supernatant to each tube of the standard curve provided by the assay and we found that this did not alter the slope of the curve (GLM: F1 = 0.81, p = 0.43; Fig. 1A). To test for parallelism we performed a twofold serial dilution of a sample pooled form eight starlings and found that the slope of this curve did not differ from the standard curve provided by the assay kit (GLM: F1 = 1.14, p = 0.36; Fig. 1B). Exogenous ACTH significantly increased FGM concentrations above saline controls (F1,14 = 28.8, p < 0.0001; Fig. 2A), and this

N.E. Cyr, L.M. Romero / General and Comparative Endocrinology 158 (2008) 20–28

Fig. 1. Assay validation (n = 8). The X-axis shows the natural log of the concentration of unlabeled GC either obtained from the kit or from the sample dilution. The Yaxis is the percent of 125I labeled GC bound (B) over that which is not bound (B0). (A) Test for accuracy. Fifty microliters of supernatant extracted from a fecal sample pooled from eight birds was added to the standard curve of the assay kit (dashed lines with open circles), and compared to the standard curve provided by the kit (black squares). (B) Test for parallelism. The supernatant of an extracted fecal sample pooled from eight birds was serially diluted (dashed lines with open circles) and compared to the standard curve of the assay kit (black squares).

increase was greatest 2 h post-injection (Fishers PLSD p < 0.0001). In contrast, exogenous progesterone did not affect FGM concentrations (Paired t7 = 0.90, p = 0.40; Fig. 2B). Although the FGM concentrations during the validation were two- to threefold higher than in the chronic stress experiment (see below), ACTH can clearly elevate FGM concentrations even with a higher baseline. 3.2. Chronic stress—captive study Estimates of total daytime plasma CORT concentrations are presented in Fig. 3. Note that because these estimates integrate responses to multiple stressors that are assumed to change over the chronic stress period (see Section 2), each of which has unknown variances, the error associated with these estimates is also unknown and therefore not presented. The average baseline GC concentration for unstressed starlings was 5.7 ng/ml (±0.94), thus we estimated the total amount of GC over the course of the 10 h day to be 57 ng/ml (Fig. 3). The response to a single administration of a 30 min acute restraint stressor was 27.8 ng/ml (±5.81); therefore, a bird exposed to 30 min of acute restraint would experience an estimated daytime GC concentration of 71 ng/ml (Fig. 3). We also found both baseline (F16,189 = 1.77, p = 0.038, not presented) and restraint-induced GC concentrations decreased during the chronic stress protocol (F16,155 = 3.77, p < 0.0001; see Fig. 3). Using

23

Fig. 2. Physiological validation (n = 8). (A) Mean FGM concentrations (±SE) preinjection and then every hour for 4 h post-injection with saline or 100 IU ACTH. (B) Mean FGM concentrations (±SE) 4 h post-injection with 5 mg/kg of progesterone or a peanut oil control. For direct comparison, we included the FGM concentration averaged over 4 h after ACTH injection (gray bar). Asterisks indicate significantly different from pre-injection using post hoc tests.

these baseline and stress-induced GC concentrations we estimated the total amount of GCs our starlings should experience each day during the chronic stress protocol as calculated from Eq. (1) (Fig. 3). The peak GC concentrations occurred on days 5 and 6 and the trough occurred on days 14–16 of the chronic stress protocol. The recovery period is not presented because we did not taken measurements from all birds and thus the samples sizes were too small. In contrast to plasma GC concentrations, FGM concentrations of unstressed, acutely stressed, and chronically stressed captive starlings did not differ during the daytime (F20,285 = 0.69, p = 0.84; Fig. 4A) or during the nighttime (F20,275 = 1.09, p = 0.36; Fig. 4B). Interestingly, we found that nighttime FGM concentrations before, during, and after the chronic stress protocol were significantly higher than daytime FGM concentrations in captive starlings (two-way repeated measures ANOVA: F20,560 = 2.32, p = 0.001). 3.3. Chronic stress—field study Adult female starlings subjected to chronic stress for 8 days had significantly higher FGM concentrations than unstressed control females (t18 = 2.35, p = 0.03; Fig. 5A). To compare nestlings in stressed and unstressed nest, nestlings were first grouped by nest. For this analysis nestlings from experimentally stressed broods had similar mean FGM concentrations to nestlings from unstressed broods (t19 = 1.15, p = 0.26; Fig. 5B). We also used a least squares model to analyze body condition (measured as mass divided by tarsus length) and treatment as predictors of FGM concentrations

24

N.E. Cyr, L.M. Romero / General and Comparative Endocrinology 158 (2008) 20–28

Fig. 3. Plasma GCs. The white bars represent estimates of plasma GC concentrations (ng/ml) integrated over the 10 h day in unstressed, acutely stressed (30 min restraint) and chronically stressed (CS) starlings. The data were calculated using baseline and restraint-induced GC data from several different studies conducted in our lab. See Section 2 for a detailed description of the calculations. Samples sizes are inside the bars and vary because not all experiments sampled all birds on all days. We do not include error bars for these results because they are estimates rather than direct measurements and thus variability is unknown. For example we use data from the GC response to restraint to deduce the response to other stressors. The black line represents the acute restraint-induced GC concentrations of acutely stressed and chronically stressed starlings. These data were taken as point samples immediately after 30 min of restraint and are used to calculate an adjustment factor for all stressors applied on that day (see Section 2).

and found that FGM concentrations negatively correlated with body condition (F1,41 = 6.37, p = 0.013; Fig. 6) and, in this analysis, nestlings in stressed nests tended to have higher FGM concentrations (F1,41 = 4.45, p = 0.042); however, nestling condition and treatment were not correlated (F1,41 = 0.11, p = 0.3). 4. Discussion Several recent reviews have highlighted the potential significance of using FGM analysis as a noninvasive measure of stress and population health as well as the importance of technical, physiological, and biological validation of FGM analysis for individual species (e.g., Goymann, 2005; Klasing, 2005; Mostl et al., 2005; Palme, 2005; Touma and Palme, 2005). Current research has demonstrated that FGM concentrations change with several factors such as diet (Klasing, 2005), gut transport time (Morrow et al., 2002), food intake and metabolic rate (Goymann et al., 2006), fluctuations in other steroid concentrations (Hunt et al., 2006) and acute stress (Wells et al., 2003). In order for FGM analysis to be a reliable indicator of population health, it is equally important to understand how FGMs change during chronic stress. The present study experimentally induced chronic stress in captive and freeliving European starlings and presents three major findings. First, FGM analysis was validated for European starlings. Second, FGM analysis did not detect any GC changes in chronically stressed captive starlings. Third, FGM concentrations were higher in chronically stressed free-living starlings compared to unstressed controls, thus supporting the assumption that chronic stress increases FGMs in wild animals. 4.1. Validation studies We used several tests to validate both the efficacy of our assay at measuring starling FGMs and the physiological accuracy of detecting GC metabolites in starling fecal samples. We found that the MP Biomedicals 125I RIA kit assay kit was successful at measur-

ing FGM concentrations in European starlings. Our tests for accuracy and parallelism did not alter the slope of the standard curve provided by the assay kit. Furthermore, ACTH injection significantly increased FGM concentrations above saline controls. The greatest increase in FGM concentrations occurred 2 h post ACTH injection indicating a 2-h time lag for GC clearance. These results are similar to those reported in other bird species. For example, ACTH caused a peak increase in FGM’s 2 h post-injection in chickens (Gallus gallus) (Rettenbacher et al., 2004) and Northern spotted owls (Wasser et al., 1997), 1 h post-injection in European stone chats (Saxicola torquata rubicola) (Goymann et al., 2002) and 3– 4 h post-injection in capercaillies (Tetrao urogallus) (Thiel et al., 2005). Studies in other avian species have reported that a similar dose of ACTH resulted in an even greater increase in FGM concentrations. In these studies, a deconjugation step was used to improve extraction. Thus, future studies should include this step (see Goymann et al., 2002). In contrast to ACTH, progesterone injection did not affect FGM concentrations of our captive starlings, which indicates that our assay is not measuring the metabolites of progesterone. Taken together, these results demonstrate that the MP Biomedicals 125I RIA kit assay kit is appropriate for measuring FGM concentrations in starlings. This was not surprising given that this assay has also been shown to accurately measure FGM concentrations in a number of mammal and bird species (Wasser et al., 2000). Interestingly, we found that mean nighttime FGM concentrations were approximately twofold higher than mean daytime FGM concentrations in our captive birds. This diurnal pattern is consistent with the 2–2½-fold increase in plasma GC concentrations found in other birds species during the early morning hours prior to activity (Breuner et al., 1999; Rich and Romero, 2001). The fact that FGM analysis was able to detect the normal diel GC rhythm further suggests the validity of using FGM analysis in starlings. However, it is also possible that the day/night comparisons are subject to differences in the time over which defecation took place (9 h during the day vs. 15 h during the night). We observed,

N.E. Cyr, L.M. Romero / General and Comparative Endocrinology 158 (2008) 20–28

25

Fig. 5. FGM concentrations—field study. Sample sizes are in the bars, and asterisks denote statistical significance compared to chronically stressed birds. (A) Mean FGM concentrations (±SE) of unstressed and chronically stressed adult female starlings. (B) Mean FGM concentrations (±SE) of nestlings from unstressed and chronically stressed broods. FGM concentrations of nestlings were averaged within each brood and then averaged across treatments.

Fig. 4. FGM concentrations—captive study (n = 16 for all days). Unstressed birds were left undisturbed, acute stressed birds experienced 30 min of restraint during the day, CS represents each day of the chronic stress protocol, and REC represents recovery or days after the completion of the chronic stress protocol. Dotted vertical lines denote the onset and completion of the chronic stress protocol. Concentrations were determined from a pool of all the fecal pellets defecated by an individual bird during that sample period and averaged over all 16 birds. (A) Mean daytime FGM concentrations (±SE) of starlings before, during, and after the chronic stress protocol. (B) Mean nighttime FGM concentrations (±SE) of starlings before, during, and after the chronic stress protocol.

but did not quantify, differences in total fecal mass during the day and night (with night being smaller), but without total fecal masses it is unclear how FGM concentrations relate to different plasma GC excretion rates. 4.2. Chronic stress—captive study Previous research has shown that baseline and acute restraintinduced plasma GC concentrations significantly attenuate with chronic stress in captive starlings (Rich and Romero, 2005). We compiled data from several earlier experiments conducted in our lab to integrate the total amount of plasma GCs that a starling subjected to normal (unstressed), acute stress, and chronic stress conditions would be predicted to experience over the course of a 10-h day (Fig. 3). We recognize that these estimates rest upon several untested assumptions, but we propose that these estimates can provide a qualitative picture of how chronic stress alters GC concentrations. In addition, a more detailed analysis would be impossible given the size of starlings. Furthermore, because all of the daytime fecal pellets were combined prior to assaying such that each sample represents the FGM concentrations over the course

Fig. 6. Relationship between nestling FGM concentrations (ng/g) and nestling condition, measured as mass over tarsus length (r2 = 0.21).

of the 10 h day, integrating the plasma GC data provides a better qualitative comparison to our FGM data than the point sample analysis performed by Rich and Romero (2005). Based on our estimates of integrated plasma GCs, we expected FGM concentrations to increase significantly on days 5–6 of the chronic stress protocol, but to decrease significantly at the end of the chronic stress protocol (Table 1 and Fig. 3). This, however, was not the case. Instead we found that mean daytime FGM concentrations did not change before, during, or after the chronic stress protocol. Mean nighttime FGM concentrations also did not change before, during, or after chronic stress. There a several potential explanations for these results. First, the stress of captivity may have masked the effects of our chronic stress protocol on FGM concentrations. Captivity has been shown

26

N.E. Cyr, L.M. Romero / General and Comparative Endocrinology 158 (2008) 20–28

Table 1 Qualitative comparison of plasma GC concentrations to FGM concentrations Treatment

Plasma GC concentrations a

FGM concentrations

ACTH (100 IU injection)

12-fold increase

Diel rhythm

2–2½ increase at nightb,c

Twofold increase at night

Chronic stress Peak increase (days 5 – 6)

Twofold increase

No change

Chronic stress End (days 15–16)

Twofold decrease

No change

a b c

Fivefold increase

Rich and Romero (2005). Breuner et al. (1999). Rich and Romero (2001).

to alter baseline and acute stress-induced plasma GC concentrations in other bird species such as white-throated sparrows (Zonotrichia albicollis), white crowned sparrows (Zonotrichia leucophrys), and lapland longspurs (Calcarius lapponicus) (Marra et al., 1995; Romero and Wingfield, 1998). Furthermore, unstressed free-living starlings in the present study had lower FGM concentrations than unstressed captive starlings (see Figs. 4 and 5). Thus, our birds may have already been chronically stressed by captivity prior to the initiation of the chronic stress protocol so that any further increases in GCs would not be detectable with FGMs. However, this seems unlikely because the estimated increase in plasma GC during days 5–6 of chronic stress was similar to the diel increase which was detected by FGM analysis. Second, chronic stress may have altered the GC clearance rate. One significant factor in GC clearance is the capacity of the GC binding protein (corticosterone binding globulin—CBG) such that the less CBG the more the liver has access to GCs in order to metabolize them (Breuner and Orchinik, 2002). If CBG capacity changed during chronic stress, thereby altering the ratio of unbound:bound GC concentrations and the clearance rate of total GCs, FGM concentrations normalized to a set time period (i.e., the 10 h of 1 day) would not be comparable. However, this is unlikely because daytime CBG capacity does not change with chronic stress in starlings (Cyr et al., 2007). In addition, we would predict that nighttime FGMs should compensate for any daytime changes in clearance rates, which was not the case in the present study. Third, FGM analysis may not be an adequate measure of subtle changes in plasma GCs during stress. Although ACTH is often used as a physiological validation to connect plasma GCs to FGMs, the dose of ACTH commonly used may stimulate a stronger GC response than endogenous release. In starlings a 100 IU injection of ACTH caused a 12-fold increase in plasma GC concentrations, but only a fivefold increase in FGM concentrations (Table 1). Even though the acute stressors used in our chronic stress protocol have been shown to increase plasma GC concentrations, several of these stressors do not produce a comparable increase in plasma GC concentrations to exogenous ACTH (Rich and Romero, 2005), and as such FGM analysis might not have been able to detect GC changes during chronic stress. However, this is also unlikely because our estimated total plasma GC concentrations take into account the different responses due to the different stressors. Fourth, our chronic stress protocol may not have been stressful. This explanation is highly unlikely given that previous studies have shown that this chronic stress protocol causes weight loss (Rich and Romero, 2005) and loss of reproductive success in starlings (Cyr and Romero, 2007). Fifth, chronic stress may have altered defecation rates, which affected FGM concentrations. Measured FGM concentrations are highly dependent upon gut transport times and defecation rates ((Bamberg et al., 2001; Morrow et al., 2002). We know starlings lose weight during the chronic stress protocol (Rich and Romero, 2005) and although we did not quantify defecation rate, there is anecdotal evidence that the birds provided fewer fecal samples

at the end of the chronic stress protocol. This could have had the effect of concentrating FGMs in the smaller fecal output, thereby artificially elevating measured concentrations at the end of the chronic stress period. If the total mass and FGM concentrations are quantified over a given period of time, the rate of excreted FGMs can be calculated and this may provide a more accurate assessment of the changes in FGM concentrations during stress (see Goymann et al., 2006). Therefore, future experiments with this chronic stress protocol should quantify total fecal output. Similarly, it is possible that the birds shunted glucocorticoid metabolites to urine, which would have cleared faster than in the feces (Bamberg et al., 2001). One way to test whether reduced food intake alters defecation rates and FGM concentrations is to include a food restricted group in subsequent chronic stress trials. 4.3. Chronic stress—field study Several studies have used FGM concentrations as a noninvasive physiological measure of stress in wild populations (e.g., Creel et al., 1997a; Foley et al., 2001; Wasser et al., 1997) and most of these studies assume that higher FGM concentrations signify stress. Increased FGM concentrations has been reported in wild animals exposed to chronic anthropogenic stressors such as logging activity (Wasser et al., 1997) and human snowmobilers (Creel et al., 2002). However, to our knowledge only one other study on black grouse (Tetrao tetrix) (Arlettaz et al., 2007) has experimentally linked an anthropogenic stressor to increases in FGM concentrations. A recent review (Romero, 2004) listed at least five possible explanations why individuals in one population would have higher GC levels than individuals in another population, only one of which is chronic stress. The present study experimentally induced chronic stress in free-living European starlings to test whether FGM concentration increase as a direct result of chronic stress. Interestingly, we found that chronically stressed adult female starlings had significantly higher FGM concentrations than unstressed starlings. These results support the assumption that higher FGM concentrations indicate chronic stress. However, these results also are inconsistent with our previously reported data on plasma GC concentrations in chronically stressed adult female starlings. Similar to laboratory studies, plasma GCs decreased after chronic stress (Cyr and Romero, 2007). A subset of birds was used in both studies, but plasma GCs and FGM concentrations did not correlate. The disparate results from these two studies likely reflect the fact that plasma GC samples are point samples whereas fecal GC measurements are more long-term measurements. Furthermore, our field results are inconsistent with our laboratory results. It is possible that the chronic stress protocol evoked a more intense GC response in the field than in the laboratory. This protocol has been shown to reduce reproductive success in freeliving starlings indicating that the chronic stress protocol may be more severe in the wild. Also, the GC response to each of the acute stressors has been tested in the lab (Nephew et al., 2003; Rich and Romero, 2005); however, the GC response to each stressor in the

N.E. Cyr, L.M. Romero / General and Comparative Endocrinology 158 (2008) 20–28

field is unknown. Future research should verify the magnitude of the GC response to each individual acute stressor in the field. Nestlings from experimentally stressed and unstressed broods had similar FGM concentrations. Cyr and Romero (2007) showed that nestlings had similar baseline GC concentrations, but nestlings from stressed nests had a greater response to acute restraint stress. Fecal samples in the present study were taken from nestlings that were not subjected to acute restraint stress. Thus, our FGM analysis supported the finding that nestlings of stressed nests do not show altered baseline GC physiology. However, FGM concentrations may reflect nestling condition given that FGM concentrations were negatively correlated with body condition in our study. 5. Conclusion FGM analysis has received much attention in the conservation literature due to its potential as a noninvasive measure of stress. The major assumption of FGM analysis has long been that higher FGM concentrations indicate stress. The present study provided empirical data demonstrating increased FGM concentrations in chronically stressed free-living starlings. These findings are consistent with the general assumption that higher FGM concentrations signify stress. However, we were unable to detect any changes in FGM concentrations in chronically stressed captive starlings. Our laboratory results are particularly disconcerting because conditions were much more controlled in the lab than in the field, which suggests that even more caution may be required for using this method in free-living animals where conditions cannot be as easily controlled. Also, one would certainly expect chronic stress to have a greater impact on GC physiology than normal daily variation; however, our FGM analysis was able to detect changes in GCs due to daily rhythms and not chronic stress in captive birds. These results have serious implications for the use of FGM analysis as a tool to assess stress in zoo animals, domestic animals, and laboratory animals. Overall we suggest that more research is needed before FGM concentrations can be used as a reliable indicator of stress. Acknowledgments We thank Jason Cyr, Molly Dickens, and Julia Wilkinson for help in the field; and Melinda Franceschini, Kristen Earle, and Rachel Olanoff for help in the lab. We also thank Dr. J.M. Reed for valuable statistical advice, and Wayne Rosky for taking excellent care of our captive starlings. This study was funded by the U.S. National Science Foundation NSF Grant: IBN-0235044 and IOB-0542099 to L.M.R., the Tufts Institute for the Environment graduate student fellowship, and the EPA Science to achieve Results fellowship: FP-91649101-0 to N.E.C. References Arlettaz, R. et al., 2007. Spreading free-riding snow sports represent a novel serious threat for wildlife. Proceedings of the Royal Society B: Biological Sciences 274, 1219–1224. Baltic, M. et al., 2005. A noninvasive technique to evaluate human-generated stress in the black grouse. Annals of the New York Academy of Sciences 1046, 81–95. Bamberg, E. et al., 2001. Excretion of corticosteroid metabolites in urine and faeces of rats. Laboratory Animals 35, 307–314. Breuner, C.W., Orchinik, M., 2002. Plasma binding proteins as mediators of corticosteroid action in vertebrates. Journal of Endocrinology 175, 99–112. Breuner, C.W. et al., 1999. Diel rhythms of basal and stress-induced corticosterone in a wild, seasonal vertebrate, Gambel’s white-crowned sparrow. Journal of Experimental Zoology 284, 334–342. Brownie, A.C., 1992. The metabolism of adrenal cortical steroids. In: James, V.H.T. (Ed.), The Adrenal Gland. Raven Press, New York, pp. 20–224. Cabe, P.R., 1993. European starling (Sturnus vulgaris). In: Gill, A.P.A.G. (Ed.), In the Birds of North America. The Academy of Natural Sciences and American Ornithologists’ Union, Philadelphia, Washington DC.

27

Creel, S. et al., 1997a. Rank and reproduction in cooperatively breeding African wild dogs: behavioral and endocrine correlates. Behavioral Ecology 8, 298– 306. Creel, S. et al., 1997b. Radiocollaring and stress hormones in African wild dogs. Conservation Biology 11, 544–548. Creel, S. et al., 2002. Snowmobile activity and glucocorticoid stress responses in wolves and elk. Conservation Biology 16, 809–814. Cyr, N.E. et al., 2007. The effect of chronic psychological stress on corticosterone, plasma metabolites, and immune responsiveness in European starlings. General and Comparative Endocrinology 154, 59–66. Cyr, N.E., Romero, L.M., 2007. Chronic stress in free-living European starlings reduces corticosterone concentrations and reproductive success. General and Comparative Endocrinology 151, 82–89. Foley, C.A.H. et al., 2001. Noninvasive stress and reproductive measures of social and ecological pressures in free-ranging African elephants. Conservation Biology 15, 1134–1142. Goymann, W., 2005. Noninvasive monitoring of hormones in bird droppings: physiological validation, sampling, extraction, sex differences, and the influence of diet on hormone metabolite levels. Annals of the New York Academy of Sciences 1046, 35–53. Goymann, W. et al., 2002. Corticosterone metabolites can be measured noninvasively in excreta of European stonechats (Saxicola torquata rubicola). Auk 119, 1167–1173. Goymann, W. et al., 2006. Low ambient temperature increases food intake and dropping production, leading to incorrect estimates of hormone metabolite concentrations in European stonechats. Hormones and Behavior 49, 644–653. Hirschenhauser, K. et al., 2005. Synthesis of measuring steroid metabolites in goose feces. Annals of the New York Academy of Sciences 1046, 138–153. Homan, R.N. et al., 2003. Impacts of varying habitat quality on the physiological stress of spotted salamanders (Ambystoma maculatum). Animal Conservation 6, 11–18. Hopkins, W.A. et al., 1997. Increased circulating levels of testosterone and corticosterone in southern toads, Bufo terrestris, exposed to coal combustion waste. General and Comparative Endocrinology 108, 237–246. Hunt, K.E. et al., 2006. Analysis of fecal glucocorticoids in the North Atlantic right whale (Eubalaena glacialis). General and Comparative Endocrinology 148, 260– 272. Klasing, K.C., 2005. Potential impact of nutritional strategy on noninvasive measurements of hormones in birds. Bird Hormones and Bird Migrations: Analyzing Hormones in Droppings and Egg Yolks and Assessing Adaptations in Long-Distance Migration. Annals of the New York Academy of Sciences 1046, 5– 16. Marra, P.P. et al., 1995. Plasma-corticosterone levels in 2 species of Zonotrichia sparrows under captive and free-living conditions. Wilson Bulletin 107, 296– 305. Millspaugh, J.J., Washburn, B.E., 2004. Use of fecal glucocorticoid metabolite measures in conservation biology research: considerations for application and interpretation. General and Comparative Endocrinology 138, 189–199. Morrow, C.J. et al., 2002. Fecal glucocorticoid metabolites as a measure of adrenal activity in dairy cattle. General and Comparative Endocrinology 126, 229–241. Mostl, E., Palme, R., 2002. Hormones as indicators of stress. Domestic Animal Endocrinology 23, 67–74. Mostl, E. et al., 2005. Measurement of corticosterone metabolites in birds’ droppings: an analytical approach. Annals of the New York Academy of Sciences 1046, 17–34. Nephew, B.C. et al., 2003. Heart rate and behavior are regulated independently of corticosterone following diverse acute stressors. General and Comparative Endocrinology 133, 173–180. Nephew, B.C., Romero, L.M., 2001. Behavioral, cardiovascular, and endocrine responses of European starlings (Sturnus vulgaris) to acute crowding. American Zoologist 41, 1538. Nephew, B.C., Romero, L.M., 2003. Behavioral, physiological, and endocrine responses of starlings to acute increases in density. Hormones and Behavior 44, 222–232. Palme, R., 2005. Measuring fecal steroids: guidelines for practical application. Annals of the New York Academy of Sciences 1046, 75–80. Remage-Healey, L., Romero, L.M., 2001. Corticosterone and insulin interact to regulate glucose and triglyceride levels during stress in a bird. American Journal of Physiology: Regulatory Integrative and Comparative Physiology 281, R994– R1003. Rettenbacher, S. et al., 2004. Measurement of corticosterone metabolites in chicken droppings. British Poultry Science 45, 704–711. Rich, E.L., Romero, L.M., 2001. Daily and photoperiod variations of basal and stressinduced corticosterone concentrations in house sparrows (Passer domesticus). Journal of Comparative Physiology B: Biochemical Systemic and Environmental Physiology 171, 543–547. Rich, E.L., Romero, L.M., 2005. Exposure to chronic stress downregulates corticosterone responses to acute stressors. American Journal of Physiology: Regulatory Integrative and Comparative Physiology 288, R1628–R1636. Romero, L.M., 2004. Physiological stress in ecology: lessons from biomedical research. Trends in Ecology & Evolution 19, 249–255. Romero, L.M., Remage-Healey, L., 2000. Daily and seasonal variation in response to stress in captive starlings (Sturnus vulgaris): corticosterone. General and Comparative Endocrinology 119, 52–59. Romero, L.M., Wingfield, J.C., 1998. Seasonal changes in adrenal sensitivity alter corticosterone levels in Gambel’s white-crowned sparrows (Zonotrichia

28

N.E. Cyr, L.M. Romero / General and Comparative Endocrinology 158 (2008) 20–28

leucophrys gambelii). Comparative Biochemistry and Physiology C: Pharmacology Toxicology & Endocrinology 119, 31–36. Sapolsky, R.M., 1992. Stress, the Ageing Brain, and Mechanisms of Neuronal Cell Death. MIT Press, Cambridge. Sapolsky, R.M. et al., 2000. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocrine Reviews 21, 55–89. Thiel, D. et al., 2005. Measuring corticosterone metabolites in droppings of capercaillies (Tetrao urogallus). Annals of the New York Academy of Sciences 1046, 96–108. Touma, C., Palme, R., 2005. Measuring fecal glucocorticoid metabolites in mammals and birds: the importance of validation. Annals of the New York Academy of Sciences 1046, 54–74. Wasser, S.K. et al., 1997. Noninvasive physiological measures of disturbance in the Northern Spotted Owl. Conservation Biology 11, 1019–1022.

Wasser, S.K., Hunt, K.E., 2005. Noninvasive measures of reproductive function and disturbance in the Barred Owl, Great Horned Owl, and Northern Spotted Owl. Annals of the New York Academy of Sciences 1046, 109–137. Wasser, S.K. et al., 2000. A generalized fecal glucocorticoid assay for use in a diverse array of nondomestic mammalian and avian species. General and Comparative Endocrinology 120, 260–275. Wells, K.M.S. et al., 2003. Effects of radio-transmitters on fecal glucocorticoid levels in captive Dickcissels. Condor 105, 805–810. Wingfield, J.C., 1994. Modulation of the adrenocortical response to stress in birds. In: Davey, R.E.P.K.G., Tobe, S.S. (Eds.), Perspectives in Comparative Endocrinology. National Research Council of Canada, Ottawa, Canada, pp. 520–528. Wingfield, J.C., Romero, L.M., 2001. Adrenocortical responses to stress and their modulation in free-living vertebrates. In: McEwen, B.S. (Ed.), Handbook of Physiology; Section 7: The Endocrine System. Oxford University Press, NY, pp. 211–234.