Rebecca K. Booth

Using DNA to track the origin of the largest ivory seizure since the 1989 trade ban

The illegal ivory trade recently intensified to the highest levels ever reported. Policing this trafficking has been hampered by the inability to reliably determine geographic origin of contraband ivory. Ivory can be smuggled across multiple international borders and along numerous trade routes, making poaching hotspots and potential trade routes difficult to identify. This fluidity also makes it difficult to refute a countrys denial of poaching problems. We extend an innovative DNA assignment method to determine the geographic origin(s) of large elephant ivory seizures. A Voronoi tessellation method is used that utilizes genetic similarities across tusks to simultaneously infer the origin of multiple samples that could have one or more common origin(s). We show that this joint analysis performs better than sample-by-sample methods in assigning sample clusters of known origin. The joint method is then used to infer the geographic origin of the largest ivory seizure since the 1989 ivory trade ban. Wildlife authorities initially suspected that this ivory came from multiple locations across forest and savanna Africa. However, we show that the ivory was entirely from savanna elephants, most probably originating from a narrow east-to-west band of southern Africa, centered on Zambia. These findings enabled law enforcement to focus their investigation to a smaller area and fewer trade routes and led to changes within the Zambian government to improve antipoaching efforts. Such outcomes demonstrate the potential of genetic analyses to help combat the expanding wildlife trade by identifying origin(s) of large seizures of contraband ivory. Broader applications to wildlife trade are discussed.

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.

Glucocorticosteroid concentrations in feces and hair of captive caribou and reindeer following adrenocorticotropic hormone challenge.

Climate change and industrial development are contributing to synchronous declines in Rangifer populations across the Arctic. Chronic stress has been implicated as a proximate factor associated with decline in free-ranging populations, but its role in Rangifer is unspecified. Analysis of glucocorticosteroid (GC) concentration in feces, and more recently in hair, is a non-invasive method for monitoring stress in wildlife. Adrenocorticotropic hormone (ACTH) released from the pituitary gland stimulates GC release from the adrenals and can be administered to reflect adrenal activation. In this study, we assessed concentrations of GC metabolites in feces and cortisol in hair of Alaskan caribou (Rangifer tarandus granti) and reindeer (R. t. tarandus) following ACTH treatment. We predicted that ACTH challenge would increase concentrations of fecal GCs, but not hair cortisol because steroid deposited into the hair shaft occurs over an extended period of time (months) and is likely insensitive to acute adrenal stimulation. Adult caribou (n=10; mean age, 6.5 years old) exhibited a peak increase in fecal GCs 8h following a 2 IU/kg dose of ACTH compared to pre-injection concentrations. In contrast, sub-adult reindeer (n=10, 0.8 years old) elicited a diminished response to the same dose. Quadrupling the dose (8 IU/kg) prolonged the fecal GC response in female reindeer, but male reindeer were unresponsive. Hair cortisol was unaffected by a single ACTH challenge. Further investigation is required to ascertain whether subspecific differences in adrenal sensitivity are attributed to age or sex differences, or historical selective pressures from semi-domestication and/or sedentary life cycle in reindeer.

Distinguishing the Impacts of Inadequate Prey and Vessel Traffic on an Endangered Killer Whale (Orcinus orca) Population

Managing endangered species often involves evaluating the relative impacts of multiple anthropogenic and ecological pressures. This challenge is particularly formidable for cetaceans, which spend the majority of their time underwater. Noninvasive physiological approaches can be especially informative in this regard. We used a combination of fecal thyroid (T3) and glucocorticoid (GC) hormone measures to assess two threats influencing the endangered southern resident killer whales (SRKW; Orcinus orca) that frequent the inland waters of British Columbia, Canada and Washington, U.S.A. Glucocorticoids increase in response to nutritional and psychological stress, whereas thyroid hormone declines in response to nutritional stress but is unaffected by psychological stress. The inadequate prey hypothesis argues that the killer whales have become prey limited due to reductions of their dominant prey, Chinook salmon (Oncorhynchus tshawytscha). The vessel impact hypothesis argues that high numbers of vessels in close proximity to the whales cause disturbance via psychological stress and/or impaired foraging ability. The GC and T3 measures supported the inadequate prey hypothesis. In particular, GC concentrations were negatively correlated with short-term changes in prey availability. Whereas, T3 concentrations varied by date and year in a manner that corresponded with more long-term prey availability. Physiological correlations with prey overshadowed any impacts of vessels since GCs were lowest during the peak in vessel abundance, which also coincided with the peak in salmon availability. Our results suggest that identification and recovery of strategic salmon populations in the SRKW diet are important to effectively promote SRKW recovery.

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.

Non-Invasive Assessment of the Interrelationships of Diet, Pregnancy Rate, Group Composition, and Physiological and Nutritional Stress of Barren-Ground Caribou in Late Winter.

The winter diet of barren-ground caribou may affect adult survival, timing of parturition, neonatal survival, and postpartum mass. We used microhistological analyses and hormone levels in feces to determine sex-specific late-winter diets, pregnancy rates, group composition, and endocrine-based measures of physiological and nutritional stress. Lichens, which are highly digestible but contain little protein, dominated the diet (> 68%) but were less prevalent in the diets of pregnant females as compared to non-pregnant females and males. The amount of lichens in the diets of pregnant females decreased at higher latitudes and as winter progressed. Pregnancy rates (82.1%, 95% CI = 76.0 – 88.1%) of adult cows were within the expected range for a declining herd, while pregnancy status was not associated with lichen abundance in the diet. Most groups (80%) were of mixed sex. Male: female ratios (62:100) were not skewed enough to affect the decline. Levels of hormones indicating nutritional stress were detected in areas of low habitat quality and at higher latitudes. Levels of hormones indicated that physiological stress was greatest for pregnant cows, which faced the increasing demands of gestation in late winter. These fecal-based measures of diet and stress provided contextual information for the potential mechanisms of the ongoing decline. Non-invasive techniques, such as monitoring diets, pregnancy rates, sex ratios and stress levels from fecal samples, will become increasingly important as monitoring tools as the industrial footprint continues to expand in the Arctic.

Modulation in Persistent Organic Pollutant Concentration and Profile by Prey Availability and Reproductive Status in Southern Resident Killer Whale Scat Samples

Persistent organic pollutants (POPs), specifically PCBs, PBDEs, and DDTs, in the marine environment are well documented, however accumulation and mobilization patterns at the top of the food-web are poorly understood. This study broadens the understanding of POPs in the endangered Southern Resident killer whale population by addressing modulation by prey availability and reproductive status, along with endocrine disrupting effects. A total of 140 killer whale scat samples collected from 54 unique whales across a 4 year sampling period (2010-2013) were analyzed for concentrations of POPs. Toxicant measures were linked to pod, age, and birth order in genotyped individuals, prey abundance using open-source test fishery data, and pregnancy status based on hormone indices from the same sample. Toxicant concentrations were highest and had the greatest potential for toxicity when prey abundance was the lowest. In addition, these toxicants were likely from endogenous lipid stores. Bioaccumulation of POPs increased with age, with the exception of presumed nulliparous females. The exceptional pattern may be explained by females experiencing unobserved neonatal loss. Transfer of POPs through mobilization of endogenous lipid stores during lactation was highest for first-borns with diminished transfer to subsequent calves. Contrary to expectation, POP concentrations did not demonstrate an associated disruption of thyroid hormone, although this association may have been masked by impacts of prey abundance on thyroid hormone concentrations. The noninvasive method for measuring POP concentrations in killer whales through scat employed in this study may improve toxicant monitoring in the marine environment and promote conservation efforts.

Population growth is limited by nutritional impacts on pregnancy success in endangered Southern Resident killer whales (Orcinus orca)

The Southern Resident killer whale population (Orcinus orca) was listed as endangered in 2005 and shows little sign of recovery. These fish eating whales feed primarily on endangered Chinook salmon. Population growth is constrained by low offspring production for the number of reproductive females in the population. Lack of prey, increased toxins and vessel disturbance have been listed as potential causes of the whale’s decline, but partitioning these pressures has been difficult. We validated and applied temporal measures of progesterone and testosterone metabolites to assess occurrence, stage and health of pregnancy from genotyped killer whale feces collected using detection dogs. Thyroid and glucocorticoid hormone metabolites were measured from these same samples to assess physiological stress. These methods enabled us to assess pregnancy occurrence and failure as well as how pregnancy success was temporally impacted by nutritional and other stressors, between 2008 and 2014. Up to 69% of all detectable pregnancies were unsuccessful; of these, up to 33% failed relatively late in gestation or immediately post-partum, when the cost is especially high. Low availability of Chinook salmon appears to be an important stressor among these fish-eating whales as well as a significant cause of late pregnancy failure, including unobserved perinatal loss. However, release of lipophilic toxicants during fat metabolism in the nutritionally deprived animals may also provide a contributor to these cumulative effects. Results point to the importance of promoting Chinook salmon recovery to enhance population growth of Southern Resident killer whales. The physiological measures used in this study can also be used to monitor the success of actions aimed at promoting adaptive management of this important apex predator to the Pacific Northwest.

Physiological implications of landscape use by free-ranging maned wolves (Chrysocyon brachyurus) in Brazil

Abstract Wide-ranging species with large spatial requirements can rarely be supported in protected areas alone, yet most species face greater threats outside reserves. The need to consider conservation of large mammals in landscape mosaics is very relevant to the Brazilian Cerrado, which is the worlds most biologically diverse and threatened savanna. The maned wolf (Chrysocyon brachyurus), South Americas largest canid, has been proposed as a flagship species for the Cerrado. The majority of the maned wolfs distribution overlaps croplands, whereas < 4% of its range overlaps with a protected area. To understand how landscape use influences maned wolf physiological health and likelihood of persistence, we collected scat samples (n = 974) from a 4,000-km2 area encompassing national park, cropland, cattle pasture, and remnant vegetation. From these samples, we measured fecal hormone metabolites of glucocorticoids (indicative of stress), thyroid hormone (indicative of nutritional status), and progesterone (indicative of reproductive health). Glucocorticoid metabolite concentrations increased with distance from natural habitat patches and during times of peak harvest activity. Thyroid hormone metabolite levels were higher in areas with more cropland, indicating good nutritional status. Progesterone metabolite levels in females were higher inside than outside the park, suggesting females residing in the park have higher reproductive success. Results indicate that maned wolves make extensive use of the landscape matrix and are able to tolerate modified agricultural fields despite being sensitive to disturbance. Ensuring their conservation for the long-term will require increased monitoring and incentivizing conservation on private lands that compose much of the Cerrado. Resumo Espécies com grandes requerimentos de área raramente podem ser protegidas unicamente em unidades de conservação, pois muitas delas enfrentam significativas ameaças fora de tais espaços. A necessidade de se considerar a conservação de mamíferos de grande porte em mosaicos na paisagem é bastante relevante no Cerrado brasileiro, que é considerado a mais rica e ameaçada savana do planeta. O lobo-guará (Chrysocyon brachyurus), maior canídeo da América do Sul, tem sido considerado como uma bandeira de conservação para o Cerrado. A maior parte da distribuição do lobo-guará é sobreposta com áreas de cultivo, sendo menos de 4% efetivamente protegida. Com o intuito de investigar como o uso da paisagem influencia a saúde fisiológica do lobo-guará e sua probabilidade de persistência, coletamos amostras de fezes em uma área de 4.000 km2, que abrangeu um parque nacional, áreas de cultivo, pastagens e remanescentes de vegetação natural. A partir dessas amostras, mensuramos os níveis de glucocorticóides (indicadores de estress), hormônio tireoidiano (indicador do estado nutricional) e progesterona (indicadora da saúde reprodutiva). A concentração de glucocorticóide foi maior com o aumento da distância de áreas nativas e também com os picos de atividade de colheita da safra regional. Os níveis de hormônio tireoidiano foram maiores em áreas com cultivos, aspecto que indica uma melhor condição alimentar. O nível de progesterona das fêmeas foi mais elevado dentro do parque do que em seu entorno, sugerindo que as fêmeas dentro da unidade podem ter maior sucesso de reprodução. Os resultados indicam que o lobo-guará faz uso extensivo da matriz de paisagem e é capaz de tolerar áreas modificadas por cultivos, embora seja sensível aos eventos de perturbação. Assegurar que a espécie seja mantida a longo prazo requer um aumento no monitoramento e no estímulo à sua proteção em propriedades privadas, que compreendem a maior parte do Cerrado.

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.