Journal of Comparative Physiology B

, Volume 185, Issue 4, pp 425–434 | Cite as

Monoterpenes as inhibitors of digestive enzymes and counter-adaptations in a specialist avian herbivore

  • Kevin D. KohlEmail author
  • Elizabeth Pitman
  • Brecken C. Robb
  • John W. Connelly
  • M. Denise Dearing
  • Jennifer Sorensen Forbey
Original Paper


Many plants produce plant secondary metabolites (PSM) that inhibit digestive enzymes of herbivores, thus limiting nutrient availability. In response, some specialist herbivores have evolved digestive enzymes that are resistant to inhibition. Monoterpenes, a class of PSMs, have not been investigated with respect to the interference of specific digestive enzymes, nor have such interactions been studied in avian herbivores. We investigated this interaction in the Greater Sage-Grouse (Phasianidae: Centrocercus urophasianus), which specializes on monoterpene-rich sagebrush species (Artemisia spp.). We first measured the monoterpene concentrations in gut contents of free-ranging sage-grouse. Next, we compared the ability of seven individual monoterpenes present in sagebrush to inhibit a protein-digesting enzyme, aminopeptidase-N. We also measured the inhibitory effects of PSM extracts from two sagebrush species. Inhibition of aminopeptidase-N in sage-grouse was compared to inhibition in chickens (Gallus gallus). We predicted that sage-grouse enzymes would retain higher activity when incubated with isolated monoterpenes or sagebrush extracts than chicken enzymes. We detected unchanged monoterpenes in the gut contents of free-ranging sage-grouse. We found that three isolated oxygenated monoterpenes (borneol, camphor, and 1,8-cineole) inhibited digestive enzymes of both bird species. Camphor and 1,8-cineole inhibited enzymes from chickens more than from sage-grouse. Extracts from both species of sagebrush had similar inhibition of chicken enzymes, but did not inhibit sage-grouse enzymes. These results suggest that specific monoterpenes may limit the protein digestibility of plant material by avian herbivores. Further, this work presents additional evidence that adaptations of digestive enzymes to plant defensive compounds may be a trait of specialist herbivores.


Aminopeptidase-N Digestive enzymes Greater sage-grouse Monoterpenes Sagebrush 



We would like to thank Dr. William Karasov, Dr. Mark Cook, and Taylor Jarmes for assistance with obtaining tissues from chickens. We also thank S. Vasilchenko, N. Wiggins, falconers T. Maechtle, D. Skinner, and H. Quade as well as Gus, the German short-haired pointer, Jack and Kenna the English setters, Grace and Bob the gyrfalcons, and Gabriel the gyrfalcon/peregrine falcon hybrid for assistance with collecting tissues in the field. This research was funded by the Idaho Department of Fish and Game, Idaho Governor’s Office for Species Conservation to J. S. F, a University of Utah Undergraduate Research Opportunities Grant to E. P, the National Science Foundation (DEB-1146194 and IOS-1258217 to J. S. F, DEB-1210094 to M. D. D and K. D. K, and DBI-1400456 to K. D. K), and Idaho INBRE Program-NIH Grant #P20 GM103408 to J.S.F., This is a contribution from Idaho Federal Aid in Wildlife Restoration Project W-160-R.


  1. Adams AS, Aylward FO, Adams SM, Erbilgin N, Aukema BH, Currie CR, Suen G, Raffa KF (2013) Mountain pine beetles colonizing historical and naive trees are associated with a bacterial community highly enriched in genes contributing to terpene metabolism. Appl Environ Microbiol 79:3468–3475CrossRefPubMedCentralPubMedGoogle Scholar
  2. Berenbaum M (1980) Adaptive significance of midgut pH in larval Lepidoptera. Am Nat 115:138–146CrossRefGoogle Scholar
  3. Birdlife International (2012) Centrocercus urophasianus. Accessed 15 Nov 2014Google Scholar
  4. Boyle R, McLean S, Foley WJ, Davies NW (1999) Comparative metabolism of dietary terpene, p-cymene, in generalist and specialist folivorous marsupials. J Chem Ecol 25:2109–2126CrossRefGoogle Scholar
  5. Bray RO, Wambolt CL, Kelsey RG (1991) Influence of sagebrush terpenoids on mule deer preference. J Chem Ecol 17:2053–2062CrossRefPubMedGoogle Scholar
  6. Bucher EH, Tamburini D, Abril A, Torres P (2003) Folivory in the white-tipped plantcutter Phytotoma rutila: seasonal variations in diet composition and quality. J Avian Biol 34:211–216CrossRefGoogle Scholar
  7. Chauhan A, Gupta S, Mahmood A (2007) Effect of tannic acid on brush border disaccharidases in mammalian intestine. Ind J Exp Biol 45:353–358Google Scholar
  8. Cheng C, Liu XW, Du FF, Li MJ, Xu F, Wang FQ, Liu Y, Li C, Sun Y (2013) Sensitive assay for measurement of volatile borneol, isoborneol, and the metabolite camphor in rat pharmacokinetic study of Borneolum (Bingpian) and Borneolum syntheticum (synthetic Bingpian). Acta Pharm Sinic 34:1337–1348CrossRefGoogle Scholar
  9. Connelly JW, Braun CE (1997) Long-term changes in Sage Grouse Centrocercus urophasianus populations in western North America. Wildl Biol 3:229–234Google Scholar
  10. Dearing MD, Mangione AM, Karasov WH (2000) Diet breadth of mammalian herbivores: nutrient versus detoxification constraints. Oecologia 123:397–405CrossRefGoogle Scholar
  11. Dearing MD, Foley WJ, McLean S (2005a) The influence of plant secondary metabolites on the nutritional ecology of herbivorous terrestrial vertebrates. Ann Rev Ecol Evol Syst 36:169–185CrossRefGoogle Scholar
  12. Dearing MD, McLister JD, Sorensen JS (2005b) Woodrat (Neotoma) herbivores maintain nitrogen balance on a low-nitrogen, high-phenolic forage, Juniperus monosperma. J Comp Physiol B 175:349–355CrossRefPubMedGoogle Scholar
  13. DeGabriel JL, Moore BD, Foley WJ, Johnson CN (2009) The effects of plant defensive chemistry on nutrient availability predict reproductive success in a mammal. Ecology 90:711–719CrossRefPubMedGoogle Scholar
  14. Durling NE, Catchpole OJ, Grey JB, Webby RF, Mitchell KA, Foo LY, Perry NB (2007) Extraction of phenolics and essential oil from dried sage (Salvia officinalis) using ethanol-water mixtures. Food Chem 101:1417–1424CrossRefGoogle Scholar
  15. Feeny PP (1969) Inhibitory effect of oakleaf tannin production on the hydrolysis of proteins by trypsin. Phytochem 8:2119–2126CrossRefGoogle Scholar
  16. Foley WJ, Lassak EV, Brophy J (1987) Digestion and absorption of Eucalyptus essential oils in greater glider (Petauroides volans) and brushtail possums (Trichosurus vulpecula). J Chem Ecol 13:2115–2130CrossRefPubMedGoogle Scholar
  17. Freeland WJ, Janzen DH (1974) Strategies in herbivory by mammals: the role of plant secondary compounds. Am Nat 108:269–289CrossRefGoogle Scholar
  18. Frye GG, Connelly JW, Musil DD, Forbey JS (2013) Phytochemistry predicts habitat selection by an avian herbivore at multiple spatial scales. Ecology 94:308–314CrossRefPubMedGoogle Scholar
  19. Howard PH (1997) Handbook of environmental fate and exposure data for organic chemicals. CRC Press, Boca RatonGoogle Scholar
  20. Huynh H, Feldt LS (1976) Estimation of the Box correction for degrees of freedom from sample data in randomised block and split-plot designs. J Educ Stat 1:69–82CrossRefGoogle Scholar
  21. Jongsma MA, Bolter C (1997) The adaptation of insects to plant protease inhibitors. J Insect Physiol 43:885–895CrossRefPubMedGoogle Scholar
  22. Jongsma MA, Bakker PL, Peters J, Bosch D, Stiekema WJ (1995) Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc Natl Acad Sci 92:8041–8045CrossRefPubMedCentralPubMedGoogle Scholar
  23. Karasov WH, Douglas AE (2013) Comparative digestive physiology. Compr Physiol 3:741–783PubMedGoogle Scholar
  24. Karasov WH, Martinez del Rio C (2007) Physiological Ecology: How Animals Process Energy, Nutrients, and Toxins. Princeton University Press, PrincetonGoogle Scholar
  25. Kelsey RG, Morris MS, Shafizadeh F (1976) The use of sesquiterpene lactones as taxonomic markers in the shrubby species of Artemisia (section tridentatae) in Montana. J Range Manage 29:502–505CrossRefGoogle Scholar
  26. Kelsey RG, Stephens JR, Shafizadeh F (1982) The chemical constituents of sagebrush foliage and their isolation. J Range Manage 35:617–622CrossRefGoogle Scholar
  27. Kohl KD, Dearing MD (2011) Induced and constitutive responses of digestive enzymes to plant toxins in an herbivorous mammal. J Exp Biol 214:4133–4140CrossRefPubMedGoogle Scholar
  28. Krzysztof C (2006) Aqueous solubility of liquid monoterpenes at 293 K and relationship with calculated log P value. Yakugaku Zasshi 126:307–309CrossRefGoogle Scholar
  29. Martin MM, Martin JS (1984) Surfactants: their role in preventing the precipitation of proteins by tannins in insect guts. Oecologia 61:342–345CrossRefGoogle Scholar
  30. McArthur C, Hagerman AE, Robbins CT (1991) Physiological strategies of mammalian herbivores against plant defenses. In: Palo RT, Robbins CT (eds) Plant defenses against mammalian herbivory. CRC Press, Boca Raton, USA, pp 103–114Google Scholar
  31. McWhorter TJ, Caviedes-Vidal E, Karasov WH (2009) The integration of digestion and osmoregulation in the avian gut. Biol Rev 84:533–565CrossRefPubMedGoogle Scholar
  32. Meynard C, López-Calleja MV, Bozinovic F, Sabat P (1999) Digestive enzymes of a small avian herbivore, the Rufous-tailed Plantcutter. Condor 101:904–907CrossRefGoogle Scholar
  33. Min BR, Barry TN, Attwood GT, McNabb WC (2003) The effect of condensed tannins on the nutrition and health of ruminants fed fresh temperate forages: a review. Anim Feed Sci Technol 106:3–19CrossRefGoogle Scholar
  34. Miyazawa M, Watanabe H, Kameoka H (1997) Inhibition of acetylcholinesterase activity by monoterpenoids with a p-menthane skeleton. J Agric Food Chem 45:677–679CrossRefGoogle Scholar
  35. Mole S, Waterman PG (1986) Tannic acid and proteolytic enzymes: enzyme inhibition or substrate deprivation? Phytochem 26:99–102CrossRefGoogle Scholar
  36. Ngugi RK, Hinds FC, Powell J (1995) Mountain big sagebrush browse decreases dry matter intake, digestibility, and nutritive quality of sheep diets. J Range Manage 48:487–492CrossRefGoogle Scholar
  37. Obst BS, Diamond JM (1989) Interspecific variation in sugar and amino acid transport by the avian cecum. J Exp Zool 252:117–126CrossRefGoogle Scholar
  38. Oh H, Hoff JE (1986) Effect of condensed grape tannins on the in vitro activity of digestive proteases and activation of their zymogens. J Food Sci 51:577–580CrossRefGoogle Scholar
  39. Oh HK, Sakai T, Jones MB, Longhurst WM (1967) Effect of various essential oils isolated from Douglas fir needles upon sheep and deer rumen microbial activity. Appl Microbiol 15:777–784PubMedCentralPubMedGoogle Scholar
  40. Oh HK, Jones MB, Longhurst WM (1968) Comparison of rumen microbial inhibition resulting from various essential oils isolated from relatively unpalatable plant species. Appl Microbiol 16:39–44PubMedCentralPubMedGoogle Scholar
  41. Perry NSL, Houghton PJ, Theobald A, Jenner P, Perry EK (2000) In-vitro inhibition of human erythrocyte acetylcholinesterase by Salvia lavandulaefolia essential oil and constituent terpenes. J Pharm Pharmacol 52:895–902CrossRefPubMedGoogle Scholar
  42. Pisani JM, Distel RA (1998) Inter- and intraspecific variations in production of spines and phenols in Prosopis caldenia and Prosopis flexuosa. J Chem Ecol 24:23–36CrossRefGoogle Scholar
  43. Remington TE, Braun CE (1985) Sage grouse food selection in winter, North Park, Colorado. J Wildl Manage 49:1055–1061CrossRefGoogle Scholar
  44. Rhoades DF (1977) The antiherbivore chemistry of Larrea. In: Mabry TJ, Hunziker JH, DiFeo DR (eds) Creosote bush: biology and chemistry of Larrea in New World deserts. Hutchinson and Ross, Stroudsberg, pp 135–175Google Scholar
  45. Ríos J, Zarco A, Mosca-Torres ME, Sabat P (2014) Dieta de Phytotoma rutila (Passeriformes: Cotingidae) en el desierto del Monte central, Argentina. Gayana (Concepción) 78:21–24CrossRefGoogle Scholar
  46. Rosina M, Romo M (2012) Reproducción y alimentación de Phytotoma raimondii, cortarrama peruana en El Gramadal, Ancash. Revista Peruana de Biología 19:167–173CrossRefGoogle Scholar
  47. Sarni-Manchado P, Canals-Bosch J-M, Mazerolles G, Cheynier V (2008) Influence of the glycosylation of human salivary proline-rich proteins on their interactions with condensed tannins. J Agric Food Chem 56:9563–9569CrossRefPubMedGoogle Scholar
  48. Savelev SU, Okello EJ, Perry EK (2004) Butyryl- and acetyl-cholinesterase inhibitory activities in essential oils of Salvia species and their constituents. Phytother Res 18:315–324CrossRefPubMedGoogle Scholar
  49. Shafizadeh F, Bhadane NR, Kelsey RG (1974) Sesquiterpene lactones of sage brush: constituents of Artemisia tripartita. Phytochem 13:669–670CrossRefGoogle Scholar
  50. Shipley LA, Forbey JS, Moore BD (2009) Revisiting the dietary niche: when is a mammalian herbivore a specialist? Integr Comp Biol 49:274–290CrossRefPubMedGoogle Scholar
  51. Shipley LA, Davis EM, Felicetti LA, McLean S, Forbey JS (2012) Mechanisms for eliminating monoterpenes in sagebrush by specialist and generalist rabbits. J Chem Ecol 38:1178–1189CrossRefPubMedGoogle Scholar
  52. Siddons RC (1972) Effect of diet on disaccharidase activity in the chick. Brit J Nutr 27:343–352CrossRefPubMedGoogle Scholar
  53. Sjöström H, Norén O, Olsen J (2002) Structure and function of aminopeptidase N. In: Langner J, Ansorge S (eds) Cellular peptidases in immune functions and diseases 2. Springer, USA, pp 25–34CrossRefGoogle Scholar
  54. Sorensen JS, Turnbull CA, Dearing MD (2004) A specialist herbivore (Neotoma stephensi) absorbs fewer plant toxins than does a generalist herbivore (Neotoma albigula). Physiol Biochem Zool 77:139–148CrossRefPubMedGoogle Scholar
  55. Stirby KD, Wambolt CL, Kelsey RG, Havstad KM (1987) Crude terpenoid influence on in vitro digestibility of sagebrush. J Range Manage 40:244–248CrossRefGoogle Scholar
  56. Tadera K, Minami Y, Takamatsu K, Matsuoka T (2006) Inhibition of alpha-glucosidase and alpha-amylase by flavonoids. J Nutr Sci Vitaminol 52:149–153CrossRefPubMedGoogle Scholar
  57. Takahashi T (2011) Flow behavior of digesta and the absorption of nutrients in the gastrointestine. J Nutr Sci Vitaminol 57:265–273CrossRefPubMedGoogle Scholar
  58. Thacker ET, Gardner DR, Messmer TA, Guttery MR, Dahlgren DK (2012) Using gas chromatography to determine winter diets of greater sage-grouse in Utah. J Wildl Manage 76:588–592CrossRefGoogle Scholar
  59. Ulappa AC, Kelsey RG, Frye GG, Rachlow LA, Shipley LA, Bond L, Pu X, Forbey JS (2014) Plant protein and secondary metabolites influence diet selection in a mammalian specialist herbivore. J Mamm 95:834–842 CrossRefGoogle Scholar
  60. Uribe S, Ramirez J, Peña A (1985) Effects of β-pinene on yeast membrane functions. J Bacteriol 161:1195–1200PubMedCentralPubMedGoogle Scholar
  61. Wallestad R, Peterson JG, Eng RL (1975) Foods of adult sage-grouse in central Montana. J Wildl Manage 39:628–630CrossRefGoogle Scholar
  62. Welch BL, McArthur ED (1981) Variation of monoterpenoid content among subspecies and accessions of Artemisia tridentata grown in a uniform garden. J Range Manage 34:380–384CrossRefGoogle Scholar
  63. Welsch CA, Lachance PA, Wasserman BP (1989) Effects of native and oxidized phenolic compounds on sucrase activity in rat brush border membrane. J Nutr 119:1737–1740PubMedGoogle Scholar
  64. Wiggins NL, McArthur C, McLean S, Boyle R (2003) Effects of two plant secondary metabolites, cineole and gallic acid, on nightly feeding patterns of the common brushtail possum. J Chem Ecol 29:1447–1464CrossRefPubMedGoogle Scholar
  65. Wilt FM, Miller GC (1992) Seasonal variation of coumarin and flavonoid concentrations in persistent leaves of Wyoming big sagebrush (Artemisia tridentata ssp. wyomingensis: Asteraceae). Biochem Syst Ecol 20:53–67CrossRefGoogle Scholar
  66. Wilt FM, Geddes JD, Tamma RV, Miller GC, Everett RL (1992) Interspecific variation of phenolic concentrations in persistent leaves among six taxa from subgenus Tridentatae of Artemisia (Asteraceae). Biochem Syst Ecol 20:41–52CrossRefGoogle Scholar
  67. Yao X, Peng Y, Xu LJ, Li L, Wu QL, Xiao PG (2011) Phytochemistry and biological studies of Lycium medicinal plants. Chem Biodivers 8:976–1010CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Kevin D. Kohl
    • 1
    Email author
  • Elizabeth Pitman
    • 1
  • Brecken C. Robb
    • 2
  • John W. Connelly
    • 3
  • M. Denise Dearing
    • 1
  • Jennifer Sorensen Forbey
    • 2
  1. 1.Department of BiologyUniversity of UtahSalt Lake CityUSA
  2. 2.Department of Biological SciencesBoise State UniversityBoiseUSA
  3. 3.Idaho Department of Fish and GameBlackfootUSA

Personalised recommendations