Sub-chronic exposure to a neonicotinoid does not affect susceptibility of larval leopard frogs to infection by trematode parasites, via either depressed cercarial performance or host immunity

  • Stacey A. RobinsonEmail author
  • M. J. Gavel
  • S. D. Richardson
  • R. J. Chlebak
  • D. Milotic
  • J. Koprivnikar
  • M. R. Forbes
Immunology and Host-Parasite Interactions - Original Paper


Little information is available on the effects of neonicotinoid insecticides on vertebrates. Previous work using amphibians found chronic exposure to some neonicotinoids had no detrimental effects on fitness-relevant traits. However, there is some evidence of more subtle effects of neonicotinoids on immune traits and evidence that other pesticides can suppress tadpole immunity resulting in elevated levels of parasitism in the exposed tadpoles. The objective of our study was to assess whether neonicotinoid exposure affected tadpole immunometrics and susceptibility to parasitic helminths. We assessed northern leopard frog tadpole (Lithobates pipiens) levels of parasitism and leukocyte profiles following exposure to environmentally relevant concentrations of clothianidin and free-living infective cercariae of a helminth parasite, an Echinostoma sp. trematode. When comparing tadpoles from controls to either 1 or 100 μg/L clothianidin treatments, we found similar measures of parasitism (i.e. prevalence, abundance and intensity of echinostome cysts) and similar leukocyte profiles. We also confirmed that clothianidin was not lethal for cercariae; however, slight reductions in swimming activity were detected at the lowest exposure concentration of 0.23 μg/L. Our results show that exposure to clothianidin during the larval amphibian stage does not affect leukocyte profiles or susceptibility to parasitism by larval trematodes in northern leopard frogs although other aspects such as length of host exposure require further study.


Amphibian Immunity Leukocytes Parasite Neonicotinoid Cercariae 



We thank F. Maisonneuve and E. Pelletier from Environment and Climate Change Canada for their help and support with the chemical analyses.

Funding information

Funding for this project was provided by Environment and Climate Change Canada (SR01-2016).

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

Statement on the welfare of animals

All applicable international, national and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted (Environment and Climate Change Canada Wildlife Eastern Animal Care Committee, SR01-2016). This article does not contain any studies with human participants performed by any of the authors.

Supplementary material

436_2019_6385_MOESM1_ESM.docx (27 kb)
ESM 1 (DOCX 27 kb)
436_2019_6385_MOESM2_ESM.xlsx (38 kb)
ESM 2 (XLSX 37 kb)


  1. Anderson JC, Dubetz C, Palace VP (2015) Neonicotinoids in the Canadian aquatic environment: a literature review on current use products with a focus on fate, exposure, and biological effects. Sci Total Environ 505:409–422. CrossRefGoogle Scholar
  2. Ballabeni P, Ward PI (1993) Local adaptation of the trematode Diplostomum phoxini to the European minnow Phoxinus phoxinus, in its second intermediate host. Funct Ecol 7:84–90CrossRefGoogle Scholar
  3. Bates D, Maechler M, Bolker B, Walker S (2015) Fitting linear mixed-effects models using lme4. J Stat Softw 67:1–48. CrossRefGoogle Scholar
  4. Beaver PC (1937) Experimental studies on Echinostoma revolutum (Froelich), a fluke from birds and mammals. Ill Biol Monogr 15:1–96Google Scholar
  5. Belden LK, Kiesecker JM (2005) Glucocorticosteroid hormone treatment of larval treefrogs increases infection by Alaria sp. trematode cercariae. J Parasitol 91:686–688. CrossRefGoogle Scholar
  6. Beldomenico PM, Begon M (2010) Disease spread, susceptibility and infection intensity: vicious circles? Trends Ecol Evol 25:21–27CrossRefGoogle Scholar
  7. 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 1223:108–119. CrossRefGoogle Scholar
  8. 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 B 367:1688–1707CrossRefGoogle Scholar
  9. Boone MD, Semlitsch RD, Little EE, Doyle MC (2007) Multiple stressors in amphibian communities: effects of chemical contamination, bullfrogs, and fish. Ecol Appl 17:291–301CrossRefGoogle Scholar
  10. Bridges CM (1999) Effects of a pesticide on tadpole activity and predator avoidance behavior. J Herpetol 33:303–306. CrossRefGoogle Scholar
  11. Brodkin MA, Madhoun H, Rameswaran M, Vatnick I (2007) Atrazine is an immune disruptor in adult northern leopard frogs (Rana pipiens). Environ Toxicol Chem 26:80–84CrossRefGoogle Scholar
  12. Brunner FS, Eizaguirre C (2016) Review: Can environmental change affect host/parasite-mediated speciation? Zoology 119:384–394. CrossRefGoogle Scholar
  13. Budischak SA, Belden LK, Hopkins WA (2009) Relative toxicity of malathion to trematode-infected and noninfected Rana palustris tadpoles. Arch Environ Contam Toxicol 56:123–128. CrossRefGoogle Scholar
  14. Bush AO, Lafferty KD, Lotz JM, Shostak AW (1997) Parasitology meets ecology on its own terms: Margolis et al. J Parasitol 83:575–583CrossRefGoogle Scholar
  15. Carey C (2000) Infectious disease and worldwide declines of amphibian populations, with comments on emerging diseases in coral reef organisms and in humans. Environ Health Perspect 108:143–150. Google Scholar
  16. Carey C, Bryant CJ (1995) Possible interrelations among environmental toxicants, amphibian development, and decline of amphibian populations. Environ Health Perspect 103:13–17. Google Scholar
  17. Christin MS, Dendron AD, Brousseau P et al (2003) Effects of agricultural pesticides on the immune system of Rana pipiens and on its resistance to parasitic infection. Environ Toxicol Chem 22:1127–1133CrossRefGoogle Scholar
  18. Coors A, Meester LD (2008) Synergistic, antagonistic and additive effects of multiple stressors: predation threat, parasitism and pesticide exposure in Daphnia magna. J Appl Ecol 45:1820–1828. CrossRefGoogle Scholar
  19. Cortes-Gomez AM, Ruiz-Agudelo CA, Valencia-Aguilar A, Ladle RJ (2015) Ecological functions of neotropical amphibians and reptiles: a review. Univ Sci 20:229–245. CrossRefGoogle Scholar
  20. Davis AK, Maerz JC (2009) Effects of larval density on hematological stress indices in salamanders. J Exp Zool Part A 311:697–704. CrossRefGoogle Scholar
  21. Davis AK, Maerz JC (2011) Assessing stress levels of captive-reared amphibians with hematological data: implications for conservation initiatives. J Herpetol 45:40–44. CrossRefGoogle Scholar
  22. Davis AK, Maney DL, Maerz JC (2008) The use of leukocyte profiles to measure stress in vertebrates: a review for ecologists. Funct Ecol 22:760–772. CrossRefGoogle Scholar
  23. Fournier DA, Skaug HJ, Ancheta J, Ianelli J, Magnusson A, Maunder MN, Nielsen A, Sibert J (2012) AD Model Builder: using automatic differentiation for statistical inference of highly parameterized complex nonlinear models. Optim Methods Softw 27:233–249. CrossRefGoogle Scholar
  24. Fox J, Weisberg S (2011) Cox proportional-hazards regression for survival data in R. In: An R companion to applied regression, second edi. Sage, Thousand Oaks, pp 1–20Google Scholar
  25. Fried B, Pane PL, Reddy A (1997) Experimental infection of Rana pipiens tadpoles with Echinostoma trivolvis cercariae. Parasitol Res 83:666–669CrossRefGoogle Scholar
  26. Gavel MJ, Richardson SD, Dalton RL, Soos C, Ashby B, McPhee L, Forbes MR, Robinson SA (2019) Effects of two neonicotinoid insecticides on blood cell profiles and corticosterone concentrations of wood frogs (Lithobates sylvaticus). Environ Toxicol Chem 38:1273–1284CrossRefGoogle Scholar
  27. Gervasi SS, Foufopoulos J (2008) Costs of plasticity: responses to desiccation decrease post-metamorphic immune function in a pond-breeding amphibian. Funct Ecol 22:100–108. Google Scholar
  28. Gilbertson M, Haffner GD, Droullard KG, Albert A, Dixon B (2003) Immunosuppression of the northern leopard frog (Rana pipiens) induced by pesticide exposure. Environ Toxicol Chem 22(1):101–110CrossRefGoogle Scholar
  29. Gosner KL (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16:183–190. Google Scholar
  30. Hadji-Azimi I, Coosemans V, Canicatti C (1987) Atlas of adult Xenopus laevis laevis hematology. Dev Comp Immunol 11:807–874. CrossRefGoogle Scholar
  31. Harris ML, Bishop CA, Struger J, Ripley B, Bogart JP (1998) The functional integrity of northern leopard frog (Rana pipiens) and green frog (Rana clamitans) populations in orchard wetlands. Effects of pesticides and eutrophic conditions on early life stage development. Environ Toxicol Chem 17:1351–1363. CrossRefGoogle Scholar
  32. Hayes TB, Case P, Chui S, Chung D, Haeffele C, Haston K, Lee M, Mai VP, Marjuoa Y, Parker J, Tsui M (2006) Pesticide mixtures, endocrine disruption, and amphibian declines: are we underestimating the impact? Environ Health Perspect 114:40–50. CrossRefGoogle Scholar
  33. Hayes TB, Falso P, Gallipeau S, Stice M (2010) The cause of global amphibian declines: a developmental endocrinologist’s perspective. J Exp Biol 213:921–933. CrossRefGoogle Scholar
  34. Hladik ML, Kolpin DW, Kuivila KM (2014) Widespread occurrence of neonicotinoid insecticides in streams in a high corn and soybean producing region, USA. Environ Pollut 193:189–196. CrossRefGoogle Scholar
  35. Hrynyk MA, Brunetti C, Kerr L, Metcalfe CD (2018) Effect of imidacloprid on the survival of Xenopus tadpoles challenged with wild type frog virus 3. Aquat Toxicol 194:152–158. CrossRefGoogle Scholar
  36. Hua J, Buss J, Kim J et al (2016) Population-specific toxicity of six insecticides to the trematode Echinoparyphium sp. Parasitology 143:542–550. CrossRefGoogle Scholar
  37. Johnson PTJ, Chase JM, Dosch KL, Hartson RB, Gross JA, Larson DJ, Sutherland DR, Carpenter SR (2007) Aquatic eutrophication promotes pathogenic infection in amphibians. Proc Natl Acad Sci 104:15781–15786. CrossRefGoogle Scholar
  38. Johnson PT, Rohr JR, Hoverman JT et al (2012) Living fast and dying of infection: host life history drives interspecific variation in infection and disease risk. Ecol Lett 15:235–242. CrossRefGoogle Scholar
  39. Jones KE, Patel NG, Levy MA, Storeygard A, Balk D, Gittleman JL, Daszak P (2008) Global trends in emerging infectious diseases. Nature 451:990–994. CrossRefGoogle Scholar
  40. 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 99:9900–9904. CrossRefGoogle Scholar
  41. Koprivnikar J (2010) Interactions of environmental stressors impact survival and development of parasitized larval amphibians. Ecol Appl 20:2263–2272. CrossRefGoogle Scholar
  42. Koprivnikar J, Walker PA (2011) Effects of the herbicide atrazine’s metabolites on host snail mortality and production of trematode cercariae. J Parasitol 97:822–827. CrossRefGoogle Scholar
  43. Koprivnikar J, Forbes MR, Baker RL (2006a) On the efficacy of anti-parasite behaviour: a case study of tadpole susceptibility to cercariae of Echinostoma trivolvis. Can J Zool 84:1623–1629CrossRefGoogle Scholar
  44. Koprivnikar J, Forbes MR, Baker RL (2006b) Effects of atrazine on cercarial longevity, activity, and infectivity. J Parasitol 92:306–311. CrossRefGoogle Scholar
  45. Koprivnikar J, Forbes MR, Baker RL (2007) Contaminant effects on host-parasite interactions: atrazine, frogs, and trematodes. Environ Toxicol Chem 26:2166–2170. CrossRefGoogle Scholar
  46. Koprivnikar J, Marcogliese DJ, Rohr JR, Orlofske SA, Raffel TR, Johnson PTJ (2012) Macroparasite infections of amphibians: what can they tell us? Ecohealth 9:342–360. CrossRefGoogle Scholar
  47. Koprivnikar J, Hoye BJ, Urichuk TM, Johnson PT (2019) Endocrine and immune responses of larval amphibians to trematode exposure. Parasitol Res 118:275–288. CrossRefGoogle Scholar
  48. Kuznetsova A, Brockhoff PB, Haubo Bojesen R (2016) lmerTest: tests in linear mixed effects models. R package version 2.0–33Google Scholar
  49. Lee-Jenkins SS, Robinson SA (2018) Effects of neonicotinoids on putative escape behaviour of juvenile wood frogs (Lithobates sylvaticus) chronically exposed as tadpoles. Environ Toxicol Chem 37:3115–3123. CrossRefGoogle Scholar
  50. Lu Z, Challis JK, Wong CS (2015) Quantum yields for direct photolysis of neonicotinoid insecticides in water: implications for exposure to non-target aquatic organisms. Environ Sci Technol Lett 2:188–192. CrossRefGoogle Scholar
  51. MacCulloch RD (2002) The ROM field guide to amphibians and reptiles of Ontario. McClelland & Stewart LtdGoogle Scholar
  52. Mann RM, Hyne RV, Choung CB, Wilson SP (2009) Amphibians and agricultural chemicals: review of the risks in a complex environment. Environ Pollut 157:2903–2927. CrossRefGoogle Scholar
  53. Marcogliese DJ, Pietrock M (2011) Combined effects of parasites and contaminants on animal health: parasites do matter. Trends Parasitol 27:123–130. CrossRefGoogle Scholar
  54. Martin TR, Conn DB (1990) The pathogenicity, localization, and cyst structure of echinostomatid metacercariae (trematode) infecting the kidneys of the frogs Rana clamitans and Rana pipiens. J Parasitol 76:414–419CrossRefGoogle Scholar
  55. Mason R, Tennekes H, Sanchez-Bayo F, Jepsen PU (2013) Immune suppression by neonicotinoid insecticides at the root of global wildlife declines. J Environ Immunol Toxicol 1:3–12. CrossRefGoogle Scholar
  56. Miles JC, Hua J, Sepulveda MS, Krupke CH, Hoverman JT (2017) Effects of clothianidin on aquatic communities: evaluating the impacts of lethal and sublethal exposure to neonicotinoids. PLoS One 12:e0174171. CrossRefGoogle Scholar
  57. Milotic D, Milotic M, Koprivnikar J (2017) Effects of road salt on larval amphibian susceptibility to parasitism through behavior and immunocompetence. Aquat Toxicol 189:42–49. CrossRefGoogle Scholar
  58. Milotic M, Milotic D, Koprivnikar J (2018) Exposure to a cyanobacterial toxin increases larval amphibian susceptibility to parasitism. Parasitol Res 117:513–520CrossRefGoogle Scholar
  59. Morrissey CA, Mineau P, Devries JH, Sanchez-Bayo F, Liess M, Cavallaro MC, Liber K (2015) Neonicotinoid contamination of global surface waters and associated risk to aquatic invertebrates: a review. Environ Int 74:291–303CrossRefGoogle Scholar
  60. Nakagawa S, Cuthill I (2007) Effect size, confidence interval and statistical significance: a practical guide for biologists. Biol Rev 82:591–605. CrossRefGoogle Scholar
  61. OECD (2009) Test no. 231: amphibian metamorphosis assay, OECD guidelines for testing of chemicals section 2: effects on biotic systems. 33Google Scholar
  62. Orlofske SA, Belden LK, Hopkins WA (2013) Larval wood frog (Rana [= Lithobates] sylvatica) development and physiology following infection with the trematode parasite, Echinostoma trivolvis. Comp Biochem Physiol A Mol Integr Physiol 164:529–536.
  63. Orlofske SA, Belden LK, Hopkins WA (2017) Effects of Echinostoma trivolvis metacercariae infection during development and metamorphosis of the wood frog (Lithobates sylvaticus). Comp Biochem Physiol A Mol Integr Physiol 203:40–48.
  64. Paetow LJ, McLaughlin JD, Cue RI et al (2012) Effects of herbicides and the chytrid fungus Batrachochytrium dendrobatidis on the health of post-metamorphic northern leopard frogs (Lithobates pipiens). Ecotoxicol Environ Saf 80:372–380. CrossRefGoogle Scholar
  65. Pietrock M, Marcogliese DJ (2003) Free-living endohelminth stages: at the mercy of environmental conditions. Trends Parasitol 19:293–299. CrossRefGoogle Scholar
  66. Pochini KM, Hoverman JT (2017a) Reciprocal effects of pesticides and pathogens on amphibian hosts: the importance of exposure order and timing. Environ Pollut 221:359–366. CrossRefGoogle Scholar
  67. Pochini KM, Hoverman JT (2017b) Immediate and lag effects of pesticide exposure on parasite resistance in larval amphibians. Parasitology 144:817–822. CrossRefGoogle Scholar
  68. Poulin R (1999) The functional importance of parasites in animal communities: many roles at many levels? Int J Parasitol 29:903–914CrossRefGoogle Scholar
  69. Prosser RS, De Solla SR, Holman EAM et al (2016) Sensitivity of the early-life stages of freshwater mollusks to neonicotinoid and butenolide insecticides. Environ Pollut 218:428–435. CrossRefGoogle Scholar
  70. Puglis HJ, Boone MD (2011) Effects of technical-grade active ingredient vs. commercial formulation of seven pesticides in the presence or absence of UV radiation on survival of green frog tadpoles. Arch Environ Contam Toxicol 60:145–155. CrossRefGoogle Scholar
  71. R Core Team (2017) R: a language and environment for statistical computing, 3.4.0. Doc. Free. available internet http//www. r-project. orgGoogle Scholar
  72. Robinson SA, Richardson SD, Dalton RL, Maisonneuve F, Trudeau VL, Pauli BD, Lee-Jenkins SSY (2017) Sublethal effects on wood frogs chronically exposed to environmentally relevant concentrations of two neonicotinoid insecticides. Environ Toxicol Chem 36:1101–1109. CrossRefGoogle Scholar
  73. Robinson SA, Richardson SD, Dalton RL, Maisonneuve F, Bartlett AJ, Solla SR, Trudeau V, Waltho N (2019) Assessment of sub-lethal effects of neonicotinoid insecticides on the life-history traits of two frog species. Environ Toxicol Chem.
  74. Rohr JR, McCoy KA (2009) A qualitative meta-analysis reveals consistent effects of atrazine on freshwater fish and amphibians. Environ Health Perspect 118:20–32. CrossRefGoogle Scholar
  75. Rohr JR, Raffel TR, Sessions SK, Hudson PJ (2008) Understanding the net effects of pesticides on amphibian trematode infections. Ecol Appl 18:1743–1753CrossRefGoogle Scholar
  76. Rohr JR, Civitello DJ, Crumrine PW, Halstead NT, Miller AD, Schotthoefer AM, Stenoien C, Johnson LB, Beasley VR (2015) Predator diversity, intraguild predation, and indirect effects drive parasite transmission. Proc Natl Acad Sci 112:3008–3013. CrossRefGoogle Scholar
  77. Rohr JR, Brown J, Battaglin WA, McMahon TA (2017) A pesticide paradox: fungicides indirectly increase fungal infections. Ecol Appl 27:2290–2302. CrossRefGoogle Scholar
  78. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675CrossRefGoogle Scholar
  79. Skaug HJ, Fournier DA, Bolker BM, et al (2016) Generalized linear mixed models using “AD Model Builder”Google Scholar
  80. Skelly DK, Bolden SR, Holland MP et al (2006) Urbanization and disease in amphibians. In: Collinge SK, Ray C (eds) Disease ecology: community structure and pathogen dynamics. Oxford University Press, New York, pp 153–167CrossRefGoogle Scholar
  81. Spolyarich N, Hyne RV, Wilson SP, Palmer CG (2011) Morphological abnormalities in frogs from a rice-growing region in NSW, Australia, with investigations into pesticide exposure. Environ Monit Assess 173:397–407. CrossRefGoogle Scholar
  82. Starner K, Goh KS (2012) Detections of the neonicotinoid insecticide imidacloprid in surface waters of three agricultural regions of California, USA, 2010-2011. Bull Environ Contam Toxicol 88:316–321. CrossRefGoogle Scholar
  83. Sures B, Nachev M, Selbach C, Marcogliese DJ (2017) Parasite responses to pollution: what we know and where we go in “environmental parasitology.”. Parasit Vectors 10:1–19. CrossRefGoogle Scholar
  84. Szuroczki D, Richardson JML (2009) The role of trematode parasites in larval anuran communities: an aquatic ecologist’s guide to the major players. Oecologia 161:371–385. CrossRefGoogle Scholar
  85. Therneau T (2015) A package for survival analysis in S, version 2.38Google Scholar
  86. Torchin ME, Byers JE, Huspeni TC (2005) Differential parasitism of native and introduced snails: placement of parasite fauna. Biol Invasions 7:885–894CrossRefGoogle Scholar
  87. Trudeau VL, Schueler FW, Navarro-Martin L, Hamilton CK, Bulaeva E, Bennett A, Fletcher W, Taylor L (2013) Efficient induction of spawning of northern leopard frogs (Lithobates pipiens) during and outside the natural breeding season. Reprod Biol Endocrinol 11:14. CrossRefGoogle Scholar
  88. USEPA (2003) EFED risk assessment for the seed treatment of clothianidn 600FS on corn and canola. 91pp. Available online at: Accessed 31 Jan 2019
  89. Venables WN, Ripley BD (2002) Modern applied statistics with S., Fourth. Springer, New YorkCrossRefGoogle Scholar
  90. Vu M, Weiler B, Trudeau VL (2017) Time- and dose-related effects of a gonadotropin-releasing hormone agonist and dopamine antagonist on reproduction in the northern leopard frog (Lithobates pipiens). Gen Comp Endocrinol 254:86–96. CrossRefGoogle Scholar
  91. Zuur AF, Ieno EN, Walker NJ, Saveliev AA, Smith GM (2009) Mixed effects models and extensions in ecology with R. Springer Science and Business Media, New YorkCrossRefGoogle Scholar
  92. Zuur AF, Ieno EN, Elphick CS (2010) A protocol for data exploration to avoid common statistical problems. Methods Ecol Evol 1:3–14. CrossRefGoogle Scholar
  93. Zuur AF, Hilbe JM, Ieno EN (2015) A beginner’s guide to GLM and GLMM with R. Highland Statistics Ltd., NewburghGoogle Scholar

Copyright information

© Crown 2019

Authors and Affiliations

  1. 1.National Wildlife Research Centre, Wildlife and Landscape Science Directorate, Environment and Climate Change CanadaOttawaCanada
  2. 2.Department of BiologyCarleton UniversityOttawaCanada
  3. 3.Department of Chemistry and BiologyRyerson UniversityTorontoCanada

Personalised recommendations