Kathleen E. Hunt

Evidence that ship noise increases stress in right whales

Baleen whales (Mysticeti) communicate using low-frequency acoustic signals. These long-wavelength sounds can be detected over hundreds of kilometres, potentially allowing contact over large distances. Low-frequency noise from large ships (20–200 Hz) overlaps acoustic signals used by baleen whales, and increased levels of underwater noise have been documented in areas with high shipping traffic. Reported responses of whales to increased noise include: habitat displacement, behavioural changes and alterations in the intensity, frequency and intervals of calls. However, it has been unclear whether exposure to noise results in physiological responses that may lead to significant consequences for individuals or populations. Here, we show that reduced ship traffic in the Bay of Fundy, Canada, following the events of 11 September 2001, resulted in a 6 dB decrease in underwater noise with a significant reduction below 150 Hz. This noise reduction was associated with decreased baseline levels of stress-related faecal hormone metabolites (glucocorticoids) in North Atlantic right whales (Eubalaena glacialis). This is the first evidence that exposure to low-frequency ship noise may be associated with chronic stress in whales, and has implications for all baleen whales in heavy ship traffic areas, and for recovery of this endangered right whale population.

Arctic spring: hormone-behavior interactions in a severe environment

Arctic breeding birds arrive on their nesting grounds in spring when weather conditions may still be extreme (low temperature, snow). The brief Arctic summer requires that they begin breeding as early as possible to take advantage of the ephemeral abundance of food to feed young. Failure to adjust to the local phenology results in drastically reduced reproductive success. Hormone-behavior adaptations may have evolved that maximize survival and reproductive success in the Arctic. It has been shown that the interrelationship of testosterone and territorial aggression, as birds arrive on the Arctic breeding grounds, varies according to species and locality. In some, territoriality is extremely brief following which birds become apparently refractory to the effects of testosterone. Others are territorial throughout the breeding season, but the dependence of these behaviors upon activation by testosterone is lost. Extensive data also indicate that Arctic birds modulate the adrenocortical response to acute stress. Secretion of corticosterone in response to a standardized capture stress protocol, used to mimic acute stress as a function of local environmental conditions, varies with the stage in the breeding cycle. Arctic breeding birds modulate the sensitivity of the adrenocortical response to acute stress at both the population and individual levels. These modulations are thought to be adaptations to allow the onset of territorial behavior and breeding in the face of potentially stressful conditions. Behavioral and physiological responses to corticosterone treatment are also diminished. A combination of these two hormone-behavior interrelationships can form important components of the proximate mechanisms by which birds, and other vertebrates, breed successfully in a severe and often capricious environment.

Effect of Long‐Term Preservation Methods on Fecal Glucocorticoid Concentrations of Grizzly Bear and African Elephant

Fecal hormone analysis has become a powerful noninvasive tool for the study of animal endocrine status and stress physiology (Graham and Brown 1997; Wasser et al. 1997a; Whitten et al. 1998; Goymann et al. 1999; Mostl et al. 1999; Foley et al. 2001; Creel et al. 2002; Lynch et al. 2002; Morrow et al. 2002). Variation in field and storage conditions makes it essential to know whether fecal steroid concentrations change with the method and duration of sample storage. Lyophilization (freeze-drying) with subsequent storage at or below 20 C is generally regarded as the most reliable method of long-term fecal hormone preservation (e.g., Wasser et al. 1988; Terio et al. 2002). However, lyophilizers are not widely available, and therefore investigators have turned to a variety of other fecal storage methods, including freezing at 20 C, preservation in ethanol, and/or drying the feces with silica, ovens, solar radiation, fires, or other drying methods (Wasser et al. 1988; Whitten et al. 1998; Foley et al. 2001; Tecot 2001). Yet, some of these preservation methods may result in significant changes in immunoreactive hormone concentrations, varying with the preservation method, species, and hormone. In this article, we report effects of several commonly used fecal preservation methods on immunoreactive glucocorticoid concentrations in feces of two species, African elephant (Loxodonta africana) and grizzly bear (Ursus arctos horribilis), over a 2-yr period. Five different preservation methods were chosen for comparison: no preservative, oven-drying at 45 C, silicadrying, 90% ethanol, and lyophilization, each with subsequent storage either at room temperature or in a 20 C freezer. Con-

Endocrine influences on parental care during a short breeding season: testosterone and male parental care in Lapland longspurs (Calcarius lapponicus)

Abstract In males of socially monogamous birds, plasma testosterone (T) typically declines to low levels during the parental phase. Studies on multiple-brooded species indicate that high T may be incompatible with high-quality paternal care. The length of the breeding season may affect the costs and benefits of high T and its effect on paternal care. We studied the effect of experimentally elevated T on paternal care in a single-brooded species with a short breeding season, the Lapland longspur (Calcarius lapponicus). We monitored T levels and parental behavior in 16 males with subcutaneous T implants, 14 males with empty implants, and 14 unimplanted males. We videotaped nests when nestlings were 2–3 days old and again at 4–5 days. T males with 2- to 3-day-old young visited nests and fed young less often than control males, and the mates of the T males compensated with elevated visits and feedings. However, when nestlings were 4–5 days old, T males visited their nests at normal rates – though feeding movements remained below normal – and T females visited and fed at normal rates. Nestling mass and nest success were similar in both groups. Overall, high T suppresses paternal care in Lapland longspur males. The partial improvement of paternal care when nestlings are older, despite high T, may be related to the short 6-week breeding season of this arctic species, and the consequently reduced benefits of sexual behavior late in the breeding season.

The adrenocortical responses to stress in snow buntings (Plectrophenax nivalis) and Lapland longspurs (Calcarius lapponicus) at Barrow, Alaska

Free-living snow buntings (Plectrophenax nivalis) and Lapland longspurs (Calcarius lapponicus) showed marked increases in circulating corticosterone up to 1 hr post-capture, indicating that their hypothalamo-pituitary-adrenal axes responded to the acute stress of handling and restraint in a manner similar to that of other vertebrates, although the magnitude of these responses was generally less. In the snow bunting there was no difference in the adrenocortical response to stress between June (breeding) and September (just prior to autumn migration). However, individual variation in the maximum corticosterone level attained during the stressful period was significantly correlated with fat score in both species. Those with greater fat depots have reduced responsiveness to stress.

Non-invasive measurement of thyroid hormone in feces of a diverse array of avian and mammalian species

We developed and validated a non-invasive thyroid hormone measure in feces of a diverse array of birds and mammals. An I(131) radiolabel ingestion study in domestic dogs coupled with High Pressure Liquid Chromatography (HPLC) analysis, showed that peak excretion in feces occurred at 24-48h post-ingestion, with I(131)-labelled thyroid hormone metabolites excreted primarily as triiodothyronine (T3) and relatively little thyroxine (T4), at all excretion times examined. The immunoreactive T3 profile across these same HPLC fractions closely corresponded with the I(131) radioactive profile. By contrast, the T4 immunoreactive profile was disproportionately high, suggesting that T4 excretion included a high percentage of T4 stores. We optimized and validated T3 and T4 extraction and assay methods in feces of wild northern spotted owls, African elephants, howler monkeys, caribou, moose, wolf, maned wolf, killer whales and Steller sea lions. We explained 99% of the variance in high and low T3 concentrations derived from species-specific sample pools, after controlling for species and the various extraction methods tested. Fecal T3 reflected nutritional deficits in two male and three female howler monkeys held in captivity for translocation from a highly degraded habitat. Results suggest that thyroid hormone can be accurately and reliably measured in feces, providing important indices for environmental physiology across a diverse array of birds and mammals.

Factors associated with fecal glucocorticoids in Alaskan brown bears (Ursus arctos horribilis).

The aims of this study were to validate a radioimmunoassay (RIA) for quantifying glucocorticoid metabolite concentrations in the feces of Alaskan brown bears (Ursus arctos horribilis) and to investigate whether any of the following factors are associated with those concentrations: the presence of humans or other bears, fishing difficulty, sex‐age class, diet, and season. We tested an established corticosterone RIA for assay sensitivity, similarity, precision, and sample matrix effects of brown bear feces, and it proved satisfactory. We collected fecal samples from brown bears along salmon‐spawning streams and assessed fecal glucocorticoid (FG) concentrations. We observed that the factors explaining the most variation in measured concentrations were date and diet type and that there was a significant interaction between the two. We did not observe a significant effect of human and bear activities or sex‐age class on FG concentrations. This study demonstrates that although FG concentrations may be assessed in brown bears, complex dietary patterns and seasonal variations must be taken into consideration in the study design in order to make inferences regarding stress.

Noninvasive measures of reproductive function and disturbance in the barred owl, great horned owl, and northern spotted owl.

Abstract: There is an urgent need for noninvasive methods to study reproduction and environmental stress in at‐risk species such as the northern spotted owl (Strix occidentalis caurina). Two related owl species (barred owl and great horned owl) were used as surrogates to validate hormone assays for fecal metabolites of progesterone, 17β‐estradiol, testosterone, and corticosterone. Infusions of radiolabeled hormones showed that the owls excreted most hormone within 6 h. Feces and urine contained roughly equal amounts of hormone, and most fecal hormone metabolites were quite polar. The testosterone and corticosterone assays in this study bound to the major excreted metabolites of these hormones, but two progesterone assays did not appreciably bind to the major progesterone metabolites. All assays showed excellent parallelism with hydrolyzed and unhydrolyzed samples and with previously dried or undried fecal samples. Thus, samples do not require hydrolysis or prior drying. Samples from a female barred owl had significantly higher fecal estrogen, lower fecal testosterone, and higher fecal estrogen/testosterone ratio than samples from two male barred owls. The fecal estrogen/testosterone ratio was the most accurate predictor of owl gender, particularly if two or more samples are available from the same individual. Fecal corticosterone metabolites also demonstrated considerable utility for wild northern spotted owls. Fecal glucocorticoid levels varied by gender and breeding stage, being highest in male northern spotted owls early in the breeding season and highest in females when nestlings were fledging. Collectively, these studies show that noninvasive fecal hormone measurements show great promise for noninvasive assessment of reproduction and stress in wild owls.

Validation of a fecal glucocorticoid assay for Steller sea lions (Eumetopias jubatus).

The Steller sea lion (Eumetopias jubatus) is listed as endangered in parts of its range and is suspected of suffering from ecological stressors that may be reflected by fecal glucocorticoid hormones. We validated a fecal glucocorticoid assay for this species with an adrenocorticotropic hormone (ACTH) challenge. Feces were collected from captive Steller sea lions (two males and two females) for 2 days before injection with ACTH, and for 4 or more days postinjection. Feces were freeze-dried, extracted with a methanol vortex method, and assayed for glucocorticoids. The assay demonstrated good parallelism and accuracy. All animals showed the expected peak of fecal glucocorticoid excretion after ACTH injection. However, the two males had higher baselines, higher peaks, and more delayed peaks than the females. Peak glucocorticoid excretion occurred at 5 and 28 h postinjection for the two females, and at 71 and 98 h for the two males. Correction for recoveries by the addition of tritiated hormones produced ACTH profiles that were virtually identical in pattern to uncorrected data, but with higher within-sample coefficients of variation. Based on these results, we conclude that this fecal glucocorticoid assay accurately reflects endogenous adrenal activity of Steller sea lions, and that recovery corrections are not necessary for this species when using the methanol vortex extraction method. More research is needed to address possible sex differences and other possible influences on fecal glucocorticoid concentrations.

Ecological Factors Affecting the Adrenocortical Response to Stress in Chestnut‐Collared and McCown’s Longspurs (Calcarius ornatus, Calcarius mccownii)

Secretion of the steroid hormone corticosterone (CORT) in response to perturbations results in behavior patterns that can maximize survival. Three commonly cited hypotheses suggest that during breeding, there are advantages associated with suppressed (CORT) secretion in (1) the sex that is most important for parental care, (2) individuals in good body condition, and (3) species with a short breeding season and limited renesting opportunities. We addressed these hypotheses in two midlatitude breeders, chestnut‐collared and McCown’s longspurs, by assessing CORT secretion over a 1‐h period of handling. These species have congeners that are exclusively arctic breeders, and this provides a unique opportunity to assess adrenocortical responsiveness both within a phylogenetic framework and across environmental variables. In both species, males and females showed similar CORT secretion patterns in response to handling, and body condition was unrelated to CORT secretion. Additionally, although these midlatitude breeders have more opportunities to attempt a renest than their arctic congeners, their hormonal response to stress was similar to that reported for their arctic congeners. We suggest that an attenuated stress response may relate to the severity of the breeding environment and a low likelihood of successful renesting, since these variables are common to both arctic and grassland habitats.