Parasitology Research

, Volume 118, Issue 1, pp 275–288 | Cite as

Endocrine and immune responses of larval amphibians to trematode exposure

  • Janet KoprivnikarEmail author
  • Bethany J. Hoye
  • Theresa M. Y. Urichuk
  • Pieter T. J. Johnson
Immunology and Host-Parasite Interactions - Original Paper


In nature, multiple waves of exposure to the same parasite are likely, making it important to understand how initial exposure or infection affects subsequent host infections, including the underlying physiological pathways involved. We tested whether experimental exposure to trematodes (Echinostoma trivolvis or Ribeiroia ondatrae) affected the stress hormone corticosterone (known to influence immunocompetence) in larvae representing five anuran species. We also examined the leukocyte profiles of seven host species after single exposure to R. ondatrae (including four species at multiple time points) and determined if parasite success differed between individuals given one or two challenges. We found strong interspecific variation among anuran species in their corticosterone levels and leukocyte profiles, and fewer R. ondatrae established in tadpoles previously challenged, consistent with defense “priming.” However, exposure to either trematode had only weak effects on our measured responses. Tadpoles exposed to E. trivolvis had decreased corticosterone levels relative to controls, whereas those exposed to R. ondatrae exhibited no change. Similarly, R. ondatrae exposure did not lead to appreciable changes in host leukocyte profiles, even after multiple challenges. Prior exposure thus influenced host susceptibility to trematodes, but was not obviously associated with shifts in leukocyte counts or corticosterone, in contrast to work with microparasites.


Disease Stress Hormone Immunity Glucocorticoids Amphibian Parasite 



We thank B. LaFonte and A. Sangiolo for assistance with experimental infections, tissue collection, and cell counts, B. Ardelli for methodological assistance, as well as J. Bowerman, J. Rohr, M. Venesky, T. Raffel, and J. Mihaljevic for providing amphibian eggs. Critical feedback useful in developing the manuscript was provided by C. Peletier and I. M. Neegan.

Funding information

This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and Canada Foundation for Innovation (JK), the National Science Foundation and National Institutes for Health (PTJJ), a fellowship from the David and Lucile Packard Foundation (PTJJ), a Rubicon fellowship from the Netherlands Organization for Scientific Research (BJH), and a NSERC Undergraduate Student Research Award (USRA) to TMYU.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed, and all procedures performed were in accordance with the ethical standards of the institution at which the studies were conducted. This article does not contain any studies with human participants performed by any of the authors.


  1. Allen JE, Maizels RM (2011) Diversity and dialogue in immunity to helminths. Nat Rev Immunol 11:375–388CrossRefGoogle Scholar
  2. Barnard CJ, Behnke JM, Gage AR, Brown H, Smithurst PR (1998) The role of parasite–induced immunodepression, rank and social environment in the modulation of behaviour and hormone concentration in male laboratory mice (Mus musculus). Proc R Soc Lond B Biol Sci 265:693–701CrossRefGoogle Scholar
  3. Belden LK, Kiesecker JM (2005) Glucocorticosteroid hormone treatment of larval treefrogs increases infection by Alaria sp. trematode cercariae. J Parasitol 91:686–688CrossRefGoogle Scholar
  4. Belden LK, Wingfield JC, Kiesecker JM (2010) Variation in the hormonal stress response among larvae of three amphibian species. J Exp Zool 313:524–531CrossRefGoogle Scholar
  5. Blaustein AR, Han BA, Relyea RA, Johnson PTJ, Buck JC, Gervasi SS, Kats LB (2011) The complexity of amphibian population declines: understanding the role of cofactors in driving amphibian losses. Ann N Y Acad Sci 1223:108–119CrossRefGoogle Scholar
  6. Blaustein AR, Gervasi SS, Johnson PTJ, Hoverman JT, Belden LK, Bradley PW, Xie GY (2012) Ecophysiology meets conservation: understanding the role of disease in amphibian population declines. Philos Trans R Soc Lond Ser B Biol Sci 367:1688–1707CrossRefGoogle Scholar
  7. Burraco P, Gomez-Mestre I (2016) Physiological stress responses in amphibian larvae to multiple stressors reveal marked anthropogenic effects even below lethal levels. Physiol Biochem Zool 89:462–472CrossRefGoogle Scholar
  8. Burraco P, Arribas R, Kulkarni SS, Buchholz DR, Gomez-Mestre I (2015) Comparing techniques for measuring corticosterone in tadpoles. Curr Zool 61:835–845CrossRefGoogle Scholar
  9. Burraco P, Miranda F, Bertó A, Vazquez LA, Gomez-Mestre I (2017) Validated flow cytometry allows rapid quantitative assessment of immune responses in amphibians. Amphibia-Reptilla 38:232–237CrossRefGoogle Scholar
  10. Cain DW, Cidlowski JA (2017) Immune regulation by glucocorticoids. Nat Rev Immunol 17:233–247CrossRefGoogle Scholar
  11. Calhoun DM, Woodhams D, Howard C, LaFonte BE, Gregory JR, Johnson PTJ (2016) Role of antimicrobial peptides in amphibian defense against trematode infection. EcoHealth 13:383–391CrossRefGoogle Scholar
  12. Carey C, Cohen N, Rollins-Smith L (1999) Amphibian declines: an immunological perspective. Dev Comp Immunol 23:459–472CrossRefGoogle Scholar
  13. Coulson PS, Wilson RA (1997) Recruitment of lymphocytes to the lung through vaccination enhances the immunity of mice exposed to irradiated schistosomes. Infect Immun 65:42–48Google Scholar
  14. Dantzer B, Fletcher QE, Boonstra R, Sheriff MJ (2014) Measures of physiological stress: a transparent or opaque window into the status, management and conservation of species? Conserv Physiol 2:cou023CrossRefGoogle Scholar
  15. Daszak P, Cunningham AA, Hyatt AD (2000) Emerging infectious diseases of wildlife-threats to biodiversity and human health. Science 287:443–449CrossRefGoogle Scholar
  16. Daszak P, Cunningham AA, Hyatt AD (2003) Infectious disease and amphibian population declines. Divers Distrib 9:141–150CrossRefGoogle Scholar
  17. Davis AK (2009) Metamorphosis-related changes in leukocyte profiles of larval bullfrogs (Rana catesbeiana). Comp Clin Pathol 18:181–186CrossRefGoogle Scholar
  18. Davis AK, Maney DL (2018) The use of glucocorticoid hormones or leucocyte profiles to measure stress in vertebrates: What’s the difference? Methods Ecol Evol 9:1556–1568CrossRefGoogle Scholar
  19. Davis AK, Maney DL, Maerz JC (2008) The use of leukocyte profiles to measure stress in vertebrates: a review for ecologists. Funct Ecol 22:760–772CrossRefGoogle Scholar
  20. Davis AK, Keel MK, Ferreira A, Maerz JC (2010) Effects of chytridiomycosis on circulating white blood cell distributions of bullfrog larvae (Rana catesbeiana). Comp Clin Pathol 19:49–55CrossRefGoogle Scholar
  21. Fast MD, Muise DM, Easy RE, Ross NW, Johnson SC (2006) The effects of Lepeophtheirus salmonis infections on the stress response and immunological status of Atlantic salmon (Salmo salar). Fish Shellfish Immunol 21:228–241CrossRefGoogle Scholar
  22. Fleming MW (1997) Cortisol as an indicator of severity of parasitic infections of Haemonchus contortus in lambs. Comp Biochem Physiol B 116:41–44CrossRefGoogle Scholar
  23. Freitas MB, Brown CT, Karasov WH (2017) Warmer temperature modifies effects of polybrominated diphenyl ethers on hormone profiles in leopard frog tadpoles (Lithobates pipiens). Environ Toxicol Chem 36:120–127CrossRefGoogle Scholar
  24. Fried B, Pane PL, Reddy A (1997) Experimental infection of Rana pipiens tadpoles with Echinostoma trivolvis cercariae. Parasitol Res 83:666–669CrossRefGoogle Scholar
  25. Fuxjager MJ, Foufopoulos J, Diaz-Uriarte R, Marler CA (2011) Functionally opposing effects of testosterone on two different types of parasite: implications for the immunocompetence handicap hypothesis. Funct Ecol 25:132–138CrossRefGoogle Scholar
  26. Gabor CR, Fisher MC, Bosch J (2015) Elevated corticosterone levels and changes in amphibian behavior are associated with Batrachochytrium dendrobatidis (Bd) infection and Bd lineage. PLoS One 10:e0122685CrossRefGoogle Scholar
  27. Gabor CR, Knutie SA, Roznik EA, Rohr JR (2018) Are the adverse effects of stressors on amphibians mediated by their effects on stress hormones? Oecologia 186:393–404CrossRefGoogle Scholar
  28. Gervasi SS, Lowry M, Hunt E, Blaustein AR (2014) Temporal patterns in immunity, infection load, and disease susceptibility: understanding the drivers of host responses in the amphibian chytrid-fungus system. Funct Ecol 28:569–578CrossRefGoogle Scholar
  29. Gosner KL (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16:183–190Google Scholar
  30. Graham AL, Cattadori IM, Lloyd-Smith JO, Ferrari MJ, Bjørnstad ON (2007) Transmission consequences of coinfection: cytokines writ large? Trends Parasitol 23:284–291CrossRefGoogle Scholar
  31. Graham AL, Shuker DM, Pollitt LC, Auld SKJR, Wilson AJ, Little TJ (2011) Fitness consequences of immune responses: strengthening the empirical framework for ecoimmunology. Funct Ecol 25:5–17CrossRefGoogle Scholar
  32. Hadji-Azimi I, Coosemans V, Canicatti C (1987) Atlas of adult Xenopus laevis laevis hematology. Dev Comp Immunol 11:807–874CrossRefGoogle Scholar
  33. Harris PD, Soleng A, Bakke TA (2000) Increased susceptibility of salmonids to the monogenean Gyrodactylus salaris following administration of hydrocortisone acetate. Parasitology 120:57–64CrossRefGoogle Scholar
  34. Harvie M, Camberis M, Tang SC, Delahunt B, Paul W, Le Gros G (2010) The lung is an important site for priming CD4 T-cell-mediated protective immunity against gastrointestinal helminth parasites. Infect Immun 78:3753–3762CrossRefGoogle Scholar
  35. Herman JP, Cullinan WE (1997) Neurocircuitry of stress: central control of the hypothalamo–pituitary–adrenocortical axis. Trends Neurosci 20:78–84CrossRefGoogle Scholar
  36. Hing S, Narayan EJ, Thompson RA, Godfrey SS (2016) The relationship between physiological stress and wildlife disease: consequences for health and conservation. Wildl Res 43:51–60CrossRefGoogle Scholar
  37. Holland MP (2009) Echinostome metacercariae cyst elimination in Rana clamitans (green frog) tadpoles is age-dependent. J Parasitol 95:281–285CrossRefGoogle Scholar
  38. Holland MP (2010) Echinostome-induced mortality varies across amphibian species in the field. J Parasitol 96:851–855CrossRefGoogle Scholar
  39. Holland MP, Skelly DK, Kashgarian M, Bolden SR, Harrison LM, Cappello M (2007) Echinostome infection in green frogs (Rana clamitans) is stage and age dependent. J Zool 271:455–462CrossRefGoogle Scholar
  40. Hoverman JT, Hoye BJ, Johnson PTJ (2013) Does timing matter? How priority effects influence the outcome of parasite interactions within hosts. Oecologia 173:1471–1480CrossRefGoogle Scholar
  41. Huver JR, Koprivnikar J, Johnson PTJ, Whyard S (2015) Development and application of an eDNA method to detect and quantify a pathogenic parasite in aquatic ecosystems. Ecol Appl 25:991–1002CrossRefGoogle Scholar
  42. Jamieson AM, Yu S, Annicelli CH, Medzhitov R (2010) Influenza virus-induced glucocorticoids compromise innate host defense against a secondary bacterial infection. Cell Host Microbe 7:103–114CrossRefGoogle Scholar
  43. Johnson PTJ, McKenzie VJ (2009) Effects of environmental change on helminth infections in amphibians: exploring the emergence of Ribeiroia and Echinostoma infections in North America. In: Fried B, Toledo R (eds) The biology of Echinostomes. Springer, New York, pp 249–280CrossRefGoogle Scholar
  44. Johnson PTJ, Kellermanns E, Bowerman J (2011) Critical windows of disease risk: amphibian pathology driven by developmental changes in host resistance and tolerance. Funct Ecol 25:726–734CrossRefGoogle Scholar
  45. Johnson PTJ, Rohr JR, Hoverman JT, Kellermanns E, Bowerman J, Lunde KB (2012) Living fast and dying of infection: host life history drives interspecific variation in infection and disease risk. Ecol Lett 15:235–242CrossRefGoogle Scholar
  46. Kiesecker JM (2002) Synergism between trematode infection and pesticide exposure: a link to amphibian limb deformities in nature? Proc Natl Acad Sci U S A 99:9900–9904CrossRefGoogle Scholar
  47. Koprivnikar J (2010) Interactions of environmental stressors impact survival and development of parasitized larval amphibians. Ecol Appl 20:2263–2272CrossRefGoogle Scholar
  48. Koprivnikar J, Marcogliese DJ, Rohr JR, Orlofske SA, Raffel TR, Johnson PTJ (2012) Macroparasite infections of amphibians: what can they tell us? EcoHealth 9:342–360CrossRefGoogle Scholar
  49. Koprivnikar J, Redfern JC, Mazier HL (2014) Variation in anti-parasite behaviour and infection among larval amphibian species. Oecologia 174:1179–1185CrossRefGoogle Scholar
  50. Korte SM, Koolhaas JM, Wingfield JC, McEwen BS (2005) The Darwinian concept of stress: benefits of allostasis and costs of allostatic load and the trade-offs in health and disease. Neurosci Biobehav Rev 29:3–38CrossRefGoogle Scholar
  51. Lafferty KD, Kuris AM (2002) Trophic strategies, animal diversity and body size. Trends Ecol Evol 17:507–513CrossRefGoogle Scholar
  52. LaFonte BE, Johnson PTJ (2013) Experimental infection dynamics: using immunosuppression and in vivo parasite tracking to understand host resistance in an amphibian–trematode system. J Exp Biol 216:3700–3708CrossRefGoogle Scholar
  53. Laidley CW, Woo PTK, Leatherland IF (1988) The stress-response of rainbow trout to experimental infection with the blood parasite Cryptobia salmositica Katz, 1951. J Fish Biol 32:253–261CrossRefGoogle Scholar
  54. Maier SF, Watkins LR (1999) Bidirectional communication between the brain and the immune system: implications for behaviour. Anim Behav 57:741–751CrossRefGoogle Scholar
  55. Maizels RM, Yazdanbakhsh M (2003) Immune regulation by helminth parasites: cellular and molecular mechanisms. Nat Rev Immunol 3:733–744CrossRefGoogle Scholar
  56. Marino JA, Holland MP, Middlemis Maher J (2014) Predators and trematode parasites jointly affect larval anuran functional traits and corticosterone levels. Oikos 123:451–460CrossRefGoogle Scholar
  57. Martin LB (2009) Stress and immunity in wild vertebrates: timing is everything. Gen Comp Endocrinol 163:70–76CrossRefGoogle Scholar
  58. Martin TR, Conn DB (1990) The pathogenicity, localization, and cyst structure of echinostomatid metacercariae (Trematoda) infecting the kidneys of the frogs Rana clamitans and Rana pipiens. J Parasitol 76:414–419CrossRefGoogle Scholar
  59. McMahon TA, Sears BF, Venesky MD et al (2014) Amphibians acquire resistance to live and dead fungus overcoming fungal immunosuppression. Nature 511:224–227CrossRefGoogle Scholar
  60. Meeusen ENT, Balic A (2000) Do eosinophils have a role in the killing of helminth parasites? Parasitol Today 16:95–101CrossRefGoogle Scholar
  61. Middlemis Maher J, Werner EE, Denver RJ (2013) Stress hormones mediate predator-induced phenotypic plasticity in amphibian tadpoles. Proc R Soc Lond B Biol Sci 280:20123075CrossRefGoogle Scholar
  62. Morales-Montor J, Newhouse E, Mohamed F, Baghdadi A, Damian RT (2001) Altered levels of hypothalamic-pituitary-adrenocortical axis hormones in baboons and mice during the course of infection with Schistosoma mansoni. J Infect Dis 183:313–320CrossRefGoogle Scholar
  63. Mougeot F, Martinez-Padilla J, Bortolotti GR, Webster MI, Piertney SB (2010) Physiological stress links parasites to carotenoid-based colour signals. J Evol Biol 23:643–650CrossRefGoogle Scholar
  64. Murdock CC, Paaijmans KP, Cox-Foster D, Read AF, Thomas MB (2012) Rethinking vector immunology: the role of environmental temperature in shaping resistance. Nat Rev Microbiol 10:869–876CrossRefGoogle Scholar
  65. Parris MJ, Cornelius TO (2004) Fungal pathogen causes competitive and developmental stress in larval amphibian communities. Ecology 85:3385–3395CrossRefGoogle Scholar
  66. Peters RH (1983) The ecological implications of body size. Cambridge University Press, CambridgeCrossRefGoogle Scholar
  67. Pettis JS, Lichtenberg EM, Andree M, Stitzinger J, Rose R (2013) Crop pollination exposes honey bees to pesticides which alters their susceptibility to the gut pathogen Nosema ceranae. PLoS One 8:e70182CrossRefGoogle Scholar
  68. Rohr JR, Raffel TR, Hall CA (2010) Developmental variation in resistance and tolerance in a multi-host–parasite system. Funct Ecol 24:1110–1121CrossRefGoogle Scholar
  69. Rollins-Smith LA (1998) Metamorphosis and the amphibian immune system. Immunol Rev 166:221–230CrossRefGoogle Scholar
  70. Rollins-Smith LA (2001) Neuroendocrine-immune system interactions in amphibians. Immunol Res 23:273–280CrossRefGoogle Scholar
  71. Rollins-Smith LA (2017) Amphibian immunity–stress, disease, and climate change. Dev Comp Immunol 66:111–119CrossRefGoogle Scholar
  72. Rollins-Smith LA, Woodhams DC (2012) Amphibian immunity: staying in tune with the environment. In: Demas GE, Nelson R (eds) Ecoimmunology. Oxford University Press, New York, pp 92–143Google Scholar
  73. Romero LM (2004) Physiological stress in ecology: lessons from biomedical research. Trends Ecol Evol 19:249–255CrossRefGoogle Scholar
  74. Roth O, Sadd BM, Schmid-Hempel P, Kurtz J (2009) Strain-specific priming of resistance in the red flour beetle, Tribolium castaneum. Proc R Soc Lond B Biol Sci 276:145–151CrossRefGoogle Scholar
  75. Saeij JP, Verburg-van Kemenade L, van Muiswinkel WB, Wiegertjes GF (2003) Daily handling stress reduces resistance of carp to Trypanoplasma borreli: in vitro modulatory effects of cortisol on leukocyte function and apoptosis. Dev Comp Immunol 27:233–245CrossRefGoogle Scholar
  76. Sapolsky RM (1993) Neuroendocrinology of the stress-response. In: Becker JB, Breedlove SM, Crews D (eds) Behavioral endocrinology. MIT Press, Cambridge, pp 287–324Google Scholar
  77. Sapolsky RM, Romero LM, Munck AU (2000) How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev 21:55–89Google Scholar
  78. Secombes CJ, Chappell LH (1996) Fish immune responses to experimental and natural infection with helminth parasites. Ann Rev Fish Dis 6:167–177Google Scholar
  79. Schoenemann KL, Bonier F (2018) Repeatability of glucocorticoid hormones in vertebrates: a meta-analysis. PeerJ 6:e4398CrossRefGoogle Scholar
  80. Schotthoefer AM, Cole RA, Beasley VR (2003a) Relationship of tadpole stage to location of echinostome cercariae encystment and the consequences for tadpole survival. J Parasitol 89:475–482CrossRefGoogle Scholar
  81. Schotthoefer AM, Koehler AV, Meteyer CU, Cole RA (2003b) Influence of Ribeiroia ondatrae (Trematoda: Digenea) infection on limb development and survival of northern leopard frogs (Rana pipiens): effects of host stage and parasite-exposure level. Can J Zool 81:1144–1153CrossRefGoogle Scholar
  82. Stoltze K, Buchmann K (2001) Effect of Gyrodactylus derjavini infections on cortisol production in rainbow trout fry. J Helminthol 75:291–294Google Scholar
  83. Sures B, Knopf K, Kloas W (2001) Induction of stress by the swimbladder nematode Anguillicola crassus in European eels, Anguilla anguilla, after repeated experimental infection. Parasitology 123:179–184Google Scholar
  84. Sutherland BJG, Koczka KW, Yasuike M, Jantzen SG, Yazawa R, Koop BF, Jones SRM (2014) Comparative transcriptomics of Atlantic Salmo salar, chum Oncorhynchus keta and pink salmon O. gorbuscha during infections with salmon lice Lepeophtheirus salmonis. BMC Genomics 15:200CrossRefGoogle Scholar
  85. Thompson RCA, Lymbery AJ, Smith A (2010) Parasites, emerging disease and wildlife conservation. Int J Parasitol 40:1163–1170CrossRefGoogle Scholar
  86. Warne RW, Crespi EJ, Brunner JL (2011) Escape from the pond: stress and developmental responses to ranavirus infection in wood frog tadpoles. Funct Ecol 25:139–146CrossRefGoogle Scholar
  87. Yeh C-M, Glöck M, Ryu S (2013) An optimized whole-body cortisol quantification method for assessing stress levels in larval zebrafish. PLoS One 8:e79406CrossRefGoogle Scholar
  88. Yehuda R, Seckl J (2011) Mini-review: stress-related psychiatric disorders with low cortisol levels: a metabolic hypothesis. Endocrinology 152:4496–4503CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Chemistry and BiologyRyerson UniversityTorontoCanada
  2. 2.Department of Ecology and Evolutionary BiologyUniversity of Colorado BoulderBoulderUSA
  3. 3.School of Biological SciencesUniversity of WollongongWollongongAustralia
  4. 4.Department of BiologyBrandon UniversityBrandonCanada

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