Development and Application of Physiological Markers of Grizzly

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Development and Application of Physiological Markers of Grizzly Bear Health David Janz and Lucy

Development and Application of Physiological Markers of Grizzly Bear Health David Janz and Lucy Kapronczai

Research question Q 4: Have changing landscape conditions associated with anthropogenic natural resource extraction

Research question Q 4: Have changing landscape conditions associated with anthropogenic natural resource extraction activities resulted in changes in habitat selection by, and the health of, grizzly bears within the study area? 2

“Conservation Medicine” The application of 21 st century technologies used in human medicine (for

“Conservation Medicine” The application of 21 st century technologies used in human medicine (for disease diagnosis, biomarker discovery, and drug development) to free-ranging wildlife species Ø “Omics” technologies (transcriptomics, proteomics, metabolomics) Ø Technological advances in analytical biochemistry (liquid chromatography coupled with tandem mass spectrometry; LCMS/MS) PHYSIOLOGY 3

Rationale Human medicine is focused on individual health, whereas wildlife biology is focused on

Rationale Human medicine is focused on individual health, whereas wildlife biology is focused on population health (i. e. , population performance and sustainability) Nevertheless, natural selection operates at the level of individual animals to maximize their fitness (Darwin, 1858) In other words, population dynamics (birth rate, mortality rate, immigration, and emigration) is controlled by the fitness of individual animals within populations PHYSIOLOGY 4

Objectives 1. Determine concentrations of hormones in hair that are associated with reproductive status

Objectives 1. Determine concentrations of hormones in hair that are associated with reproductive status (testosterone, progesterone, and estradiol) and long -term stress (cortisol) 2. Develop a novel proteomics technique to quantify expression of proteins in skin that are associated with energetics, reproduction, immune function, and physiological stress PHYSIOLOGY 5

Objective 1: Hair Hormones Similar to drugs and heavy metals, steroid hormones accumulate in

Objective 1: Hair Hormones Similar to drugs and heavy metals, steroid hormones accumulate in the hair shaft during hair growth and represent a long-term “chronology” of circulating hormone over months The grizzly bear annual hair cycle is ideal for this work, since hair grows from late spring to hibernation and thus represents much of the active season PHYSIOLOGY 6

Objective 1: Progress Techniques to determine cortisol, testosterone, progesterone and estradiol have been validated

Objective 1: Progress Techniques to determine cortisol, testosterone, progesterone and estradiol have been validated in our laboratory and in our work with captive grizzly bears Lab analyses of these hormones is ongoing as we receive hair samples from field personnel Application of the hair hormone analyses will be integrated with other theme data in the project (environmental, nutritional, and behavioural variables) PHYSIOLOGY 7

Objective 2: Skin Proteomics Emerging research field of “cutaneous neuroendocrinology” PHYSIOLOGY 8

Objective 2: Skin Proteomics Emerging research field of “cutaneous neuroendocrinology” PHYSIOLOGY 8

Objective 2: Experimental Approach Candidate proteins for the proteomics technique: Markers of energetics (energy

Objective 2: Experimental Approach Candidate proteins for the proteomics technique: Markers of energetics (energy metabolism, nutritional status): leptin, ghrelin, adiponectin, apolipoprotein Markers of reproductive status (sex, pregnancy, maturity) : relaxin, prolactin, gonadotropins, progesterone receptor Markers of immune function (immunosuppression, infection): haptoglobin, α 2 -macroglobulin, kininogen, other immunoglobulins Markers of physiological stress (responses to external stressors): glucocorticoid receptor, adrenocorticotropic hormone, corticotropin-releasing hormone, HSP 70 PHYSIOLOGY 9

Objective 2: Technique Mass spectrometry-based multiple reaction monitoring (MRM) Steps: 1. Select target proteins

Objective 2: Technique Mass spectrometry-based multiple reaction monitoring (MRM) Steps: 1. Select target proteins 2. Select 3 -5 unique (“proteotypic”) peptides 3. Select specific peptide fragment ions (“transitions”) 4. Validate using stable isotope-labeled synthetic peptides 5. Quantitation of proteins and analysis of data PHYSIOLOGY 10

Objective 2: Progress Developed technique to isolate and purify sufficient protein from small (<

Objective 2: Progress Developed technique to isolate and purify sufficient protein from small (< 0. 1 g) skin biopsies to use in MRM assay Conducted a “discovery run” using orbitrap LC-MS/MS, which identified amino acid sequences of >250 proteins in grizzly bear skin Our current work is refining the actual proteins to be included in the MRM assay The challenge is that very little amino acid sequence information is available for Ursus arctos proteins; exact amino acid sequences are ideal for developing a MRM assay PHYSIOLOGY 11

Objective 2: Progress To inform us on amino acid sequences of grizzly bear proteins,

Objective 2: Progress To inform us on amino acid sequences of grizzly bear proteins, we ran a grizzly bear skin sample on an Illumina Next. Seq benchtop sequencer We performed an “exome run”, which provided us with nucleotide base (A, T, G, C) sequences of all expressed genes in bear skin PHYSIOLOGY 12

Objective 2: Progress Using available bioinformatics software, these m. RNA sequences can then be

Objective 2: Progress Using available bioinformatics software, these m. RNA sequences can then be analyzed (aligned or “mapped”) to determine amino acid sequences of proteins expressed in skin The challenge in bioinformatics is “big data” – about 400 million alignable reads were obtained! PHYSIOLOGY 13

Applications for Resource Managers Key point: the data generated from hair hormone and skin

Applications for Resource Managers Key point: the data generated from hair hormone and skin protein analyses should be viewed as tools to assist with identifying the physiological status of individual grizzly bears These data must be used in combination with other environmental and ecological variables on a larger scale to field validate their utility as physiological markers Ø For hair cortisol concentration, we have a rich long-term dataset from 1999 Ø For other hair hormones and proteomics, data are just beginning to be collected PHYSIOLOGY 14

Applications for Resource Managers Caveat: we may not identify a “magic bullet”! That is,

Applications for Resource Managers Caveat: we may not identify a “magic bullet”! That is, in the short term we will likely not identify a single marker that indicates compromised health status Nevertheless, the strength of this work is that by determining a range of markers indicative of important physiological functions, greater insight into bear health can be obtained PHYSIOLOGY 15

Thanks for Listening! Questions? David M. Janz, Ph. D. Professor, Veterinary Biomedical Sciences University

Thanks for Listening! Questions? David M. Janz, Ph. D. Professor, Veterinary Biomedical Sciences University of Saskatchewan david. janz@usask. ca 306 -966 -7434 http: //www. usask. ca/toxicology/people/faculty/david-janz. php https: //www. usask. ca/wcvm_people/profiles/Janz_David. php PHYSIOLOGY 16