Lisa S. Hayward

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.

Japanese quail selected for high plasma corticosterone response deposit high levels of corticosterone in their eggs.

Poor habitat quality or body condition often correlates with high responsiveness of the hypothalamo‐pituitary‐adrenal (HPA) axis rather than with elevated baseline levels of glucocorticoids. We hypothesized that, for egg‐laying vertebrates, high responsiveness of the HPA axis would correspond to high concentrations of corticosterone in yolk. We tested the prediction that Japanese quail (Coturnix coturnix japonica) selected for high plasma corticosterone response to brief immobilization (HS quail) would lay eggs with higher yolk corticosterone concentrations than birds selected for low response (LS quail). Quail from both lines were left undisturbed, outside of the stressors associated with daily management, before a first round of egg collection. In a second experiment, quail of both lines were experimentally stressed during the week before egg collection. In both cases we found quail from the HS line to lay eggs with significantly higher yolk corticosterone concentrations than quail of the LS line. After exposure to added experimental stressors, the line difference was more pronounced (increasing from 62% to 96%). There was no line difference in concentrations of yolk testosterone. Our results suggest that (1) genetic differences underly differences in the transfer of maternal corticosterone to yolk and (2) females may be able to control deposition of corticosterone into yolk through a mechanism independent of baseline corticosterone titers.

Impacts of acute and long‐term vehicle exposure on physiology and reproductive success of the northern spotted owl

Stress physiologists posit that multiple simultaneous demands faced by an organism may have non-additive effects on the magnitude of their response to disturbance. The environmental assessment literature emphasizes a similar phenomenon at the population level, arguing that populations can compensate for perturbations up to a threshold, beyond which disturbance impacts may be greatly magnified—and even cause system collapse. We integrated these two approaches to examine the roles of environment, life history stage, prior disturbance experience, and their interactions on vulnerability to disturbance in a free-living species. Specifically, we examined the effects of off-highway vehicle use on the federally threatened northern spotted owl (NSO), Strix occidentalis caurina, by measuring fecal glucocorticoid metabolites (fGCs), which reflect disturbance; fecal thyroid hormone metabolites (fT3), which reflect nutrition; and the number of offspring fledged within a season. We experimentally applied one hour of mo...

Eliminating the artificial effect of sample mass on avian fecal hormone metabolite concentration

Avian endocrinology is a productive field that could benefit from increased application of non-invasive techniques. Although assay protocols vary, most studies that measure hormone metabolites in avian feces struggle with an artificial effect of sample mass on steroid metabolite concentration. Hormone metabolite concentrations measured in small samples are consistently higher than concentrations in larger samples, and this appears to be due to multiple methodological problems. We systematically tested several causal hypotheses for the mass effect. Based on results from these tests, we modified and validated our assay protocol to effectively eliminate the mass effect. Future studies should implement the following procedures when measuring hormone metabolites from small fecal samples (particularly of birds and reptiles): (1) remove urates from the fecal sample as completely as possible; (2) lyophilize the sample prior to extraction; (3) maximize accuracy of small mass measurements; (4) increase the volume of ethanol in the extraction to 15 ml per 0.05-0.1g of dried feces; and (5) eliminate ethanol from all samples prior to radioimmunoassay by drying down extract solutions and rehydrating in buffer. By applying these precautions we successfully eliminated the mass effect from fecal samples ranging in mass from 0.001 to 0.1 g using a radioimmunoassay commonly employed for studies of fecal glucocorticoid metabolites. These corrections also resulted in a more than 3-fold increase in effect size in glucocorticoid concentrations from a controlled test of the effects of 1h motorcycle exposure on northern spotted owls. These methods have important implications not only for avian studies, but for any study measuring hormone metabolites from small fecal samples.

Using Detection Dogs to Conduct Simultaneous Surveys of Northern Spotted (Strix occidentalis caurina) and Barred Owls (Strix varia)

State and federal actions to conserve northern spotted owl (Strix occidentalis caurina) habitat are largely initiated by establishing habitat occupancy. Northern spotted owl occupancy is typically assessed by eliciting their response to simulated conspecific vocalizations. However, proximity of barred owls (Strix varia)–a significant threat to northern spotted owls–can suppress northern spotted owl responsiveness to vocalization surveys and hence their probability of detection. We developed a survey method to simultaneously detect both species that does not require vocalization. Detection dogs (Canis familiaris) located owl pellets accumulated under roost sites, within search areas selected using habitat association maps. We compared success of detection dog surveys to vocalization surveys slightly modified from the U.S. Fish and Wildlife Service’s Draft 2010 Survey Protocol. Seventeen 2 km ×2 km polygons were each surveyed multiple times in an area where northern spotted owls were known to nest prior to 1997 and barred owl density was thought to be low. Mitochondrial DNA was used to confirm species from pellets detected by dogs. Spotted owl and barred owl detection probabilities were significantly higher for dog than vocalization surveys. For spotted owls, this difference increased with number of site visits. Cumulative detection probabilities of northern spotted owls were 29% after session 1, 62% after session 2, and 87% after session 3 for dog surveys, compared to 25% after session 1, increasing to 59% by session 6 for vocalization surveys. Mean detection probability for barred owls was 20.1% for dog surveys and 7.3% for vocal surveys. Results suggest that detection dog surveys can complement vocalization surveys by providing a reliable method for establishing occupancy of both northern spotted and barred owl without requiring owl vocalization. This helps meet objectives of Recovery Actions 24 and 25 of the Revised Recovery Plan for the Northern Spotted Owl.

Patterns of yolk testosterone deposition in two populations of Arctic-breeding Redpolls

The levels of maternal androgens in egg yolk vary considerably within and among clutches. In most species studied to date, high levels have been found to increase offspring competitive ability. We measured yolk testosterone in two populations of Redpolls (Carduelis flammea/hornemanni) nesting around Toolik Lake Field Station and in Barrow, Alaska to test three predictions about the patterns of yolk testosterone concentrations. First, we predicted yolk testosterone to increase with laying sequence so as to minimize the deleterious effects of hatching asynchrony on chicks from later hatched eggs in a species without delayed incubation. Second, we predicted yolk testosterone to be higher in Barrow where food is more abundant and Redpoll body condition is higher. Third, we predicted yolk testosterone to correlate positively with egg mass and yolk mass, which are two other indicators of maternal investment. An initial analysis that did not consider egg and yolk mass lent support to the first of these predictions. However, more thorough statistical analysis revealed that relationships between yolk testosterone and laying sequence and between yolk testosterone and site were not direct but rather driven by associations among yolk testosterone, egg mass, and yolk mass. While yolk testosterone concentration itself did not differ between sites, the relationship between yolk testosterone and egg and yolk mass did. Yolk testosterone correlated positively with egg mass at Barrow and showed a significant quadratic association with yolk mass in Toolik. While laying sequence and yolk testosterone appeared to be correlated when egg and yolk mass were not considered, the full statistical model suggests that egg and yolk mass are directly associated with laying sequence while yolk testosterone concentrations are not. These results suggest complicated relationships among egg mass, yolk mass, yolk testosterone, site, and laying sequence that should be taken into account in future studies. They also suggest that deposition of yolk testosterone may be a regulated rather than being a purely passive process.ZusammenfassungTestosteroneinlagerung im Eigelb bei zwei in der Arktis brütenden Birkenzeisigpopulationen Mütterliche Androgeneinlagerungen im Dotter variieren deutlich innerhalb und zwischen einzelnen Bruten. Eine höhere Konzentration von mütterlichen Androgenen im Dotter erhöht die Wettbewerbsfähigkeit in den meisten untersuchten Vogelarten. Wir haben Testosteron im Dotter in zwei Birkenzeisigpopulationen (Carduelis flammea/hornemanni) die in der Nähre der Toolik Lake Feldstation und in Barrow, Alaska brüteten, gemessen, um drei Vorhersagen über die Muster von Testosteronkonzentrationen im Dotter zu testen. Erstens erwarteten wir, dass Testosteron im Dotter mit der Legesequenz zunimmt, um schädliche Auswirkungen von Schlupf-Asynchronie später gelegter Eier in einer Art ohne verzögerter Bebrütung zu reduzieren. Zweitens erwarteten wir, dass Testosteron im Dotter in Barrow höher sein würde, da dort Nahrung in größeren Mengen verfügbar ist und Birkenzeisige dort in dementsprechend besserer Kondition waren. Drittens erwarteten wir, dass die Testosteronkonzentration im Dotter positiv mit Ei- und Dottergewicht korreliert, beides weitere Indikatoren für mütterliche Investitionen. Eine erste Analyse, die Ei- und Dottergewicht nicht berücksichtigte, lieferte Ergebnisse, die für die ersten beiden Hypothesen sprachen. Eine gründlichere statistische Analyse ergab jedoch, dass Testosteron im Dotter und Legeabfolge und Brutgebiet nicht direkt, sondern indirekt durch Assoziationen zwischen Testosteron, Ei- und Dottergewicht erzeugt wurden. Die Testosteronkonzentration im Dotter unterschied sich nicht zwischen beiden Untersuchungsgebieten, die Interaktion zwischen Testosteron und Ei- und Dottergewicht jedoch schon. In Barrow war Testosteron im Dotter signifikant positiv mit dem Eigewicht assoziiert, während für Toolik ein signifikant quadratisches Verhältnis mit Dottergewicht gefunden wurde. Das vollständige statistische Model suggeriert, dass Ei- und Dottergewicht direkt mit der Legereihefolge assoziiert sind, die Testosteronkonzentration jedoch nicht. Unsere Untersuchung legt eher komplizierte Beziehungen zwischen Ei- und Dottermasse, Testosteron im Dotter und Brutgebiet nahe. Unsere Ergebnisse haben zur Folge, dass in künftigen Studien Testosteroneinlagerung im Dotter als ein regulierter Prozess anstelle eines passiven Vorgangs gesehen werden sollte.

The design of experimental studies of noise impacts on wildlife: Issues and outcomes

Historically, most studies exposing terrestrial wildlife to anthropogenic noise experimentally have failed to prove biologically‐significant impact. These counter‐intuitive outcomes have given rise to political debate, fueled by marginal effects or weak trends in some studies that might have proved to be significant with better sampling procedures or controls. A number of recent studies have attempted to address these weaknesses by developing adequate sampling designs and balancing exposures with respect to important environmental factors (e.g., habitat type). Examples include studies of: (1) Effects of low‐flying NATO aircraft on Mexican spotted owl territory occupancy and reproduction; (2) effects of U.S. Marine Corps. helicopters on passerine reproductive success; (3) effects of operations on a U.S. Air Force bombing range on small mammal abundance; (4) impact of off‐highway vehicles on reproduction of northern goshawks in the Plumas National Forest; and (5) impact of Enduro motorcycle races on norther...