The influence of polyphenols on sage-grouse dietary preferences.

Abstract

Background

Hana L. Hoang 1, Marcella R. Fremgen 2, Jennifer S. Forbey 2, Carolyn Y. Dadabay 1

1Chemistry Department, College of Idaho, Caldwell, ID 836052Department of Biological Sciences, Boise State University, Boise, ID 83725

Greater Sage-grouse (Centrocercus urophasianus), an endangered species candidate, occupies sagebrush habitats of the western United States.  The birds feed mainly on sagebrush, which in turn produces high quantities of polyphenolic compounds to serve as a defense mechanism against these and other herbivores. Sage-grouse browse on three-tip sagebrush (Artemisia tripartia) and Wyoming big sagebrush (Artemisia tridenta ssp. wyomingensis) in proportion to their availability. However, we hypothesize that another factor in dietary preference may be differences in polyphenol contents.  A Folin-Ciocalteu colorometric assay was used to determine total polyphenol contents, and high performance liquid chromatography (HPLC) was used to compare types of individual polyphenols. Total polyphenol levels were 37% higher in Wyoming big sagebrush than in three-tip sagebrush (T-test, P<0.001, n=97). Total polyphenol levels were also 14% higher in non-browsed than browsed foliage in Wyoming big sagebrush (T-test, P=0.007, n=68), but showed no differences in three-tip sagebrush (T-test, P=0.280, n=28).   HPLC chromatograms showed preliminary differences in the specific types of polyphenols in the two sagebrush species, and between browsed and non-browsed foliage within the same species.  Better knowledge of sage-grouse dietary preferences can aid in habitat conservation. In addition, bioactive polyphenols identified in this study are candidates for new drug development.

Greater Sage-Grouse (Centrocerous urophasianus)- Mainly feeds on sagebrush, other soft plants and insects. (1)- Inhabits the sagebrush county in the western United State and southwestern Canadian provinces. (2)- Endangered species candidate due to habitat loss.Sagebrush (Artemisia sp.)- Dominant in the sage-steppe ecosystem.- Supports vast wildlife communities.- Filled with bioactive compounds: flavonols, flavones and coumarins. Polyphenols: - Family of aromatic compounds produced in plants (3)- Secondary defense mechanism to deter herbivores.- Influence sage-grouse forage selection affect habitat selection

during winter (4)- Very few animals regularly consume sagebrush

MethodsSagebrush collection:- Radio telemetry flushed birds used foot prints and pellets to identify feeding zones looked for bite marks- Collect foliage of 3 browsed plants and 3 non-browsed (within 1 meter of browsed plants)Polyphenol extraction: - Ground foliage was extracted with 90% methanol, 9.5% distilled water and 0.5% acetic acid.Total polyphenol- Folin-Ciocalteu assay:- Gallic Acid standards (1µg/µL)- Folin-Ciocalteu’s phenol reagentHigh Performance Liquid Chromatography (HPLC):- Methanol extracts of plants were analyzed using HPLC system.- Mobile phase: Solvent A- 0.01% formic acid, solvent B- 0.01% formic

acid + 99.9% acetonitrile- Phenolic compounds were identified by comparing retention times

and UV VIS spectra.

Results and Discussion- Total polyphenol levels were 37% higher in Wyoming big sagebrush than

in three-tip sagebrush (T-test, P<0.001, n=96), and were 14% higher in non-browsed than browsed foliage Wyoming big sagebrush (T-test, P=0.007, n=68),

- HPLC chromatograms showed differences in the specific types and concentrations of polyphenols both between the two sagebrush species, as well as between browsed and non-browsed foliage within the same species (preliminary results). 

- There was no significant differences between browsed and non-browsed three-tip sagebrush (T-test, P=0.280, n=28).  

- HPLC chromatograms showed only small differences between browsed and non-browsed in three-tip sagebrush (preliminary results).

Significant of the study: - Knowing what plants sage-grouse prefer to eat will help reseeding

efforts and habitat conservation- Bioactive polyphenols identified in this study are candidates for new

drug development.

Future Direction: - Quantitate and identify individual compounds using HPLC/LCMS data.

Frye, Granham. Sage-Grouse Bite-mark. [Photograph].

Objective: Compare the total polyphenol content and individual compounds between Wyoming big sagebrush (Artemisia tridentata ssp. wyomingensis) and three-tip sagebrush (Artemisia tripartia)and how it affects sage-grouse dietary preferences.

Summers, Mark. Greater Sage-Grouse. [Photograph]

Fremgen, Marcella. Sagebrush. [Photograph]

Structure of phenolic compounds. [Figure]. Retrieved from http://tilia.zf.mendelu.cz/ustavy/553/dzi/www/data/13_anti.pdf

References1. McWilliams, J. (2002). U.S. Department of Agriculture, Forest Service,

Rocky Mountain Research Station2. Sage-Grouse and Sagebrush Conservation. (2015, August 12). U.S.

DEPARTMENT OF THE INTERIOR: http://www.blm.gov/wo/st/en/prog/more/sagegrouse.html

3. American Chemical Society. (2012). Journal of Agricultural and Food Chemistry, 8773-8775.

4. Doherty, K.E., Naugle, D.E., Walker, B.L., Graham, M.J. 2008. Greater sage-grouse winter habitat selection and energy development. Journal of Wildfire Management; 72(1):187-195.

Acknowledgement- Special thanks to Dr. Carolyn Dadabay, Dr. Jenifer Forbey, Marcella Fremgen, and other lab members: Lauren James, Sophia Nosworthy, Deborah Conner.- This project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant #P20GM103408 to CYD and JSF. andNational Science Foundation DEB-1146194 and IOS-1258217, Idaho Dept Fish and Game, and BLM to JSF.

Polyphenols levels were higher in Wyoming big sagebrush than three-tip sagebrush

Figure 1: The polyphenol levels in Wyoming big sagebrush and three-tip sagebrush. T-test, P<0.001, n=96.

Figure 2: Representative HPLC chromatogram of Wyoming big sagebrush and three-tip sagebrush. X-axis is retention time, and y-axis is the absorbance (nm). Each peak represent a compound.

Polyphenols were higher in non-browsed than browsed Wyoming big sagebrush

Figure 2: The polyphenol levels in non-browsed and browsed Wyoming big sagebrush. T-test, P= 0.007, n=68.

Figure 3: Representative HPLC chromatogram of non-browsed and browsed Wyoming big sagebrush. X-axis is retention time, and y-axis is the absorbance (nm). Each peak represent a compound.

Polyphenols were higher in non-browsed than browsed three-tip sagebrush

Figure 5: The polyphenol levels in non-browsed and browsed three-tip sagebrush. T-test, P= 0.280, n=28.

Figure 6: Representative HPLC chromatogram of non-browsed and browsed three-tip sagebrush. X-axis is retention time, and y-axis is the absorbance (nm). Each peak represent a compound.

Three-tip

Wyoming Big

Non-browsed

Browsed

Non-browsed

Browsed

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*- observed distinct compounds between two variables.

Three-tip

Wyoming Big

Non-browsed

Browsed

Non-browsed

Browsed