Laryngeal Demasculinization in Wild Cane Toads Varies with Land Use

Abstract

Anthropogenic factors, including the spread of endocrine-disrupting chemicals, have been linked to alterations in the reproductive physiology, morphology, and behavior of wildlife. Few studies of endocrine disruption, however, focus on secondary sexual traits that affect mating signals, despite their importance for reproductive success. The larynx of many anurans (frogs and toads), for example, is larger in males than in females and is crucial for producing mating calls. We aim to determine if wild populations of cane toads (Rhinella marina) near sugarcane fields in Florida have demasculinized larynges when compared to populations near urban areas. We find evidence of demasculinization in both primary and secondary sexual traits in male toads living near sugarcane. Relative to body size, the laryngeal mass, vocal cord length, and dilator muscle width are all reduced in males from sugarcane regions compared to their urban counterparts. Strong correlations between primary and secondary male sexual traits indicate that demasculinization occurs in concert both within and across diverse organs, including the testes, larynx, and skin. Our results show that anurans near sugarcane fields have demasculinized reproductive systems, that this disruption extends to secondary sexual traits like the larynx, and that it is likely due to anthropogenic causes.

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Data Availability

All data generated or analyzed during this study are included in the electronic supplementary materials.

References

  1. Andersson M (1994) Sexual Selection, Princeton, NJ: Princeton University Press

    Google Scholar 

  2. Bates D, Maechler M, Bolker B, Walker S (2015) Fitting linear mixed-effects models using lme4. Journal of Statistical Software 67(1):1–48. https://doi.org/10.18637/jss.v067.i01

    Article  Google Scholar 

  3. Bókony V, Üveges B, Ujhegyi N, Verebélyi V, Nemesházi E, Csíkvári O, Hettyey A (2018) Endocrine disruptors in breeding ponds and reproductive health of toads in agricultural, urban, and natural landscapes. Science of the Total Environment 634:1335–1345. https://doi.org/10.1016/j.scitotenv.2018.03.363

    CAS  Article  PubMed  Google Scholar 

  4. Boutilier RG, Donohoe PH, Tattersall GJ, West TG (1997) Hypometabolic homeostasis in overwintering aquatic amphibians. The Journal of Experimental Biology 200:387–400

    CAS  PubMed  Google Scholar 

  5. Bridges CM, Semlitsch RD (2000) Variation in pesticide tolerance of tadpoles among and within species of Ranidae and patterns of amphibian decline. Conservation Biology 14(5):1490–1499. https://doi.org/10.1046/j.1523-1739.2000.99343.x

    Article  Google Scholar 

  6. Caliman FA, Gavrilescu M (2009) Pharmaceuticals, personal care products and endocrine disrupting agents in the environment—a review. Clean 37:277–303. https://doi.org/10.1002/clen.200900038

    CAS  Article  Google Scholar 

  7. Canty A, Ripley B (2017) boot: Bootstrap R (S-Plus) Functions. R package version 1.3-20.

  8. Carr JA, Gentles A, Smith EE, Goleman WL, Urquidi LJ, Thuett K, Kendall RJ, Giesy JP, Gross TS, Solomon KR, Van Der Kraak G (2003) Response of larval Xenopus laevis to atrazine: Assessment of growth, metamorphosis, and gonadal and laryngeal morphology. Environmental Toxicology and Chemistry 22(2):396–405. https://doi.org/10.1002/etc.5620220222

    CAS  Article  PubMed  Google Scholar 

  9. Coady KK, Murphy MB, Villeneuve DL, Hecker M, Jones PD, Carr JA, Solomon KR, Smith EE, Van Der Kraak G, Kendall RJ, Giesy JP (2005) Effects of atrazine on metamorphosis, growth, laryngeal and gonadal development, aromatase activity, and sex steroid concentrations in Xenopus laevis. Ecotoxicology and Environmental Safety 62:160–173. https://doi.org/10.1016/j.ecoenv.2004.10.010

    CAS  Article  PubMed  Google Scholar 

  10. Colafrancesco KC, Gridi-Papp M (2016) Vocal sound production and acoustic communication in amphibians and reptiles. In: Vertebrate Sound Production and Acoustic Communication, Suthers RA, Fitch WT, Fay RR, Popper AN (editors), New York, NY: Springer, pp 83–117

    Google Scholar 

  11. Edwards TM, Moore BC, Guillette LJ (2006) Reproductive dysgenesis in wildlife: A comparative view. International Journal of Andrology 29:109–121. https://doi.org/10.1111/j.1365-2605.2005.00631.x

    Article  PubMed  Google Scholar 

  12. Egea-Serrano A, Relyea RA, Tejedo M, Torralva M (2012) Understanding of the impact of chemicals on amphibians: A meta-analytic review. Ecology and Evolution 2(7):1382–1397. https://doi.org/10.1002/ece3.249

    Article  PubMed  PubMed Central  Google Scholar 

  13. Fitzpatrick LC (1976) Life history patterns of storage and utilization of lipids for energy in amphibians. Integrative and Comparative Biology 16:725–732. https://doi.org/10.1093/icb/16.4.725

    CAS  Article  Google Scholar 

  14. Gerhardt HC, Huber F (2002) Acoustic Communication in Insects and Anurans: Common Problems and Diverse Solutions, Chicago, IL: University of Chicago Press

    Google Scholar 

  15. Gingras B, Boeckle M, Herbst CT, Fitch WT (2013) Call acoustics reflect body size across four clades of anurans. Journal of Zoology 289:143–150. https://doi.org/10.1111/j.1469-7998.2012.00973.x

    Article  Google Scholar 

  16. Girish S, Saidapur SK (2000) Interrelationship between food availability, fat body, and ovarian cycles in the frog, Rana tigrina, with a discussion of the role of fat body in anuran reproduction. Journal of Experimental Zoology 286(5):487–493. https://doi.org/10.1002/(sici)1097-010x(20000401)286:5%3c487::aid-jez6%3e3.0.co;2-z

    CAS  Article  PubMed  Google Scholar 

  17. Gunderson MP, Veldhoen N, Skirrow RC, Macnab MK, Ding W, van Aggelen G, Helbing CC (2011) Effect of low dose exposure to the herbicide atrazine and its metabolite on cytochrome P450 aromatase and steroidogenic factor-1 mRNA levels in the brain of premetamorphic bullfrog tadpoles (Rana catesbeiana). Aquatic Toxicology 102:31–38. https://doi.org/10.1016/j.aquatox.2010.12.019

    CAS  Article  PubMed  Google Scholar 

  18. Guerra MA, Ryan MJ, Cannatella DC (2014) Ontogeny of sexual dimorphism in the larynx of the túngara frog, Physalaemus pustulosus. Copeia 2014(1):123–129. https://doi.org/10.1643/cg-13-051

    Article  Google Scholar 

  19. Hamlin HJ, Guillette LJ (2010) Birth defects in wildlife: the role of environmental contaminants as inducers of reproductive and developmental dysfunction. Systems Biology in Reproductive Medicine 56:113–121. https://doi.org/10.3109/19396360903244598

    CAS  Article  PubMed  Google Scholar 

  20. Harrell FE, Dupont C et al. (2018) Hmisc: Harrell Miscellaneous. R package version 4.1-1

  21. Harrison PTC, Holmes P, Humfrey CDN (1997) Reproductive health in humans and wildlife: Are adverse trends associated with environmental chemical exposure? Science of the Total Environment 205:97–106. https://doi.org/10.1016/s0048-9697(97)00212-x

    CAS  Article  PubMed  Google Scholar 

  22. Hayes TB, Collins A, Lee M, Mendoza M, Noriega N, Stuart AA, Vonk A (2002) Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Proceedings of the National Academy of Sciences of the USA 99(8):5476–5480. https://doi.org/10.1073/pnas.082121499

    CAS  Article  PubMed  Google Scholar 

  23. Hayes TB, Khoury V, Narayan A, Nazir M, Park A, Brown T, Adame L, Chan E, Buchholz D, Stueve T, Gallipeau S (2010) Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis). Proceedings of the National Academy of Sciences of the USA 107(10):4612–4617. https://doi.org/10.1073/pnas.0909519107

    Article  PubMed  Google Scholar 

  24. Hoffman RS, Capel PD, Larson SJ (2000) Comparison of pesticides in Eight U.S. Urban Streams. Environmental Toxicology and Chemistry 19(9):2249–2258. https://doi.org/10.1897/1551-5028(2000)019%3c2249:copieu%3e2.3.co;2

    CAS  Article  Google Scholar 

  25. Hoffmann F, Kloas W (2010) An environmentally relevant endocrine-disrupting antiandrogen, vinclozolin, affects calling behavior of male Xenopus laevis. Hormones and Behavior 58(4):653–659. https://doi.org/10.1016/j.yhbeh.2010.06.008

    CAS  Article  PubMed  Google Scholar 

  26. Hoffmann F, Kloas W (2012) Estrogens can disrupt amphibian mating behavior. PLoS ONE 7(2):1–8. https://doi.org/10.1371/journal.pone.0032097

    CAS  Article  Google Scholar 

  27. Hoskins TD, Boone MD (2018) Atrazine feminizes sex ratio in Blanchard’s cricket frogs (Acris blanchardi) at concentrations as low as 0.1 μg/L. Environmental Toxicology and Chemistry 37(2):427–435. https://doi.org/10.1002/etc.3962

    CAS  Article  PubMed  Google Scholar 

  28. Kloas W, Urbatzka R, Opitz R, Würtz S, Behrends T, Hermelink B, Hofmann F, Jagnytsch O, Kroupova H, Lorenz C, Neumann N, Pietsch C, Trubiroha A, Van Ballegooy C, Wiedemann C, Lutz I (2009) Endocrine disruption in aquatic vertebrates. Annals of the New York Academy of Sciences 1163:187–200. https://doi.org/10.1111/j.1749-6632.2009.04453.x

    CAS  Article  PubMed  Google Scholar 

  29. Koo TK, Li MY (2016) A guideline of selecting and reporting intraclass correlation coefficients for reliability research. Journal of Chiropractic Medicine 15:155–163. https://doi.org/10.1016/j.jcm.2016.02.012

    Article  PubMed  PubMed Central  Google Scholar 

  30. Langerveld AJ, Celestine R, Zaya R, Mihalko D, Ide CF (2009) Chronic exposure to high levels of atrazine alters expression of genes that regulate immune and growth-related functions in developing Xenopus laevis tadpoles. Environmental Research 109:379–389. https://doi.org/10.1016/j.envres.2009.01.006

    CAS  Article  PubMed  Google Scholar 

  31. Length R (2018) Emmeans: Estimated Marginal Means, aka Least-Squares Means. R package version 1.1

  32. Mackenzie CA, Berrill M, Metcalfe C, Pauli BD (2003) Gonadal differentiation in frogs exposed to estrogenic and antiestrogenic compounds. Environmental Toxicology and Chemistry 22(10):2466–2475. https://doi.org/10.1897/02-173

    CAS  Article  PubMed  Google Scholar 

  33. Mann RM, Hyne RV, Choung CB, Wilson SP (2009) Amphibians and agricultural chemicals: Review of the risks in a complex environment. Environmental Pollution 157:2903–2927. https://doi.org/10.1016/j.envpol.2009.05.015

    CAS  Article  PubMed  Google Scholar 

  34. Martin WF (1971) Mechanics of sound production in toads of the genus Bufo: Passive elements. Journal of Experimental Zoology 176(3):273–293. https://doi.org/10.1002/jez.1401760304

    CAS  Article  PubMed  Google Scholar 

  35. McClelland BE, Wilczynski W (1989) Sexually dimorphic laryngeal morphology in Rana pipiens. Journal of Morphology 201:293–299. https://doi.org/10.1002/jmor.1052010308

    CAS  Article  PubMed  Google Scholar 

  36. McCoy KA, Bortnick LJ, Campbell CM, Hamlin HJ, Guillette LJ, St. Mary CM (2008) Agriculture alters gonadal form and function in the toad Bufo marinus. Environmental Health Perspectives 116(11):1526–1532. https://doi.org/10.1289/ehp.11536

    Article  PubMed  PubMed Central  Google Scholar 

  37. McCoy KA, Hoang LK, Guillette LJ, St. Mary CM (2008b) Renal pathologies in giant toads (Bufo marinus) vary with land use. Science of the Total Environment 407:348–357. https://doi.org/10.1016/j.scitotenv.2008.09.008

    CAS  Article  PubMed  Google Scholar 

  38. McCoy KA, Amato CM, Guillette LJ, St. Mary CM (2017) Giant toads (Rhinella marina) living in agricultural areas have altered spermatogenesis. Science of the Total Environment 609:1230–1237. https://doi.org/10.1016/j.scitotenv.2017.07.185

    CAS  Article  PubMed  Google Scholar 

  39. Meshaka WE, Butterfield BP, Hauge JB (2004) The Exotic Amphibians and Reptiles of Florida, Malabar, FL: Krieger Publishing Company

    Google Scholar 

  40. Miles CJ, Pfeuffer RJ (1997) Pesticides in canals of South Florida. Archives of Environmental Contamination and Toxicology 32:337–345. https://doi.org/10.1007/s00244900194

    CAS  Article  PubMed  Google Scholar 

  41. Mossler M (2008) Florida Crop/Pest Profile: Sugarcane. UF/IFAS Extension PI-171:1-14

  42. Muller BJ, Schwarzkopf L (2017) Success of capture of toads improved by manipulating acoustic characteristics of lures. Pest Management Science 73:2372–2378. https://doi.org/10.1002/ps.4629

    CAS  Article  PubMed  Google Scholar 

  43. Orton F, Baynes A, Clare F, Duffus ALJ, Larroze S, Scholze M, Garner TWJ (2014) Body size, nuptial pad size and hormone levels: Potential non-destructive biomarkers of reproductive health in wild toads (Bufo bufo). Ecotoxicology 23:1359–1365. https://doi.org/10.1007/s10646-014-1261-3

    CAS  Article  PubMed  Google Scholar 

  44. Orton F, Tyler CR (2015) Do hormone-modulating chemicals impact on reproduction and development of wild amphibians? Biological Reviews 90(4):1100–1117. https://doi.org/10.1111/brv.12147

    Article  PubMed  Google Scholar 

  45. Pattersson I, Berg C (2007) Environmentally relevant concentrations of ethynylestradiol cause female-biased sex ratios in Xenopus tropicalis and Rana temporaria. Environmental Toxicology and Chemistry 26(5):1005–1009. https://doi.org/10.1897/06-464r.1

    Article  Google Scholar 

  46. Piprek RP, Kloc M, Kubiak JZ (2014) Bidder’s organ—Structure, development and function. International Journal of Developmental Biology 58:819–827. https://doi.org/10.1387/ijdb.140147rp

    CAS  Article  PubMed  Google Scholar 

  47. Qin ZF, Qin XF, Yan L, Han-Ting L, Zhao XR, Xu XB (2007) Feminizing/demasculinizing effects of polychlorinated biphenyls on the secondary sexual development of Xenopus laevis. Aquatic Toxicology 84:321–327. https://doi.org/10.1016/j.aquatox.2007.06.011

    CAS  Article  PubMed  Google Scholar 

  48. R Core Team (2017) R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing

    Google Scholar 

  49. Regnault C, Usal M, Veyrenc S, Couturier K, Batandier C, Bulteau AL, Lejon D, Sapin A, Combourieu B, Chetiveaux M, Le May C, Lafond T, Raveton M, Reynaud S (2018) Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline. Proceedings of the National Academy of Sciences of the USA 115(19):E4416–E4425. https://doi.org/10.1073/pnas.1721267115

    CAS  Article  PubMed  Google Scholar 

  50. Ryan MJ (2001) Anuran Communication, Washington, DC: Smithsonian Institution Press

    Google Scholar 

  51. Sassoon D, Kelley DB (1986) The sexually dimorphic larynx of Xenopus laevis: Development and androgen regulation. American Journal of Anatomy 177:457–472. https://doi.org/10.1002/aja.1001770404

    CAS  Article  PubMed  Google Scholar 

  52. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9(7):671–675. https://doi.org/10.1038/nmeth.2089

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. Schug TT, Johnson AF, Birnbaum LS, Colborn T, Guillette LJ, Crews DP, Collins T, Soto AM, vom Saal FS, McLachlan JA, Sonnenschein C, Heindel JJ (2016) Minireview: Endocrine disruptors: Past lessons and future directions. Molecular Endocrinology 30(8):833–847. https://doi.org/10.1210/me.2016-1096

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. Sitja-Bobadilla A (2009) Can myxosporean parasites compromise fish and amphibian reproduction? Proceedings of the Royal Society B 276:2861–2870. https://doi.org/10.1098/rspb.2009.0368

    Article  PubMed  Google Scholar 

  55. Solomon KR, Carr JA, Du Preez LH, Giesy JP, Kendall RJ, Smith EE, Van Der Kraak GJ (2008) Effects of atrazine on fish, amphibians, and aquatic reptiles: A critical review. Critical Reviews in Toxicology 38(9):721–772. https://doi.org/10.1080/10408440802116496

    Article  PubMed  Google Scholar 

  56. Smith EE, Du Preez LH, Gentles A, Solomon KR, Tandler B, Carr JA, Van Der Kraak GL, Kendall RJ, Giesy JP, Gross TS (2005) Assessment of laryngeal muscle and testicular cell types in Xenopus laevis (Anura: Pipidae) inhabiting maize and non-maize growing areas of South Africa. African Journal of Herpetology 54(1):69–76. https://doi.org/10.1080/21564574.2005.9635519

    Article  Google Scholar 

  57. Tamschick S, Rozenblut-Koscisty B, Ogielska M, Lehmann A, Lymberakis P, Hoffmann F, Lutz I, Kloas W, Stock M (2016) Sex reversal assessments reveal different vulnerability to endocrine disruption between deeply diverged anuran lineages. Scientific Reports 6:23825. https://doi.org/10.1038/srep23825

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  58. Tattersall GJ, Ultsch GR (2008) Physiological ecology of aquatic overwintering in ranid frogs. Biological Reviews 83:119–140. https://doi.org/10.1111/j.1469-185x.2008.00035.x

    Article  PubMed  Google Scholar 

  59. Wake DB, Vredenburg VT (2008) Are we in the midst of the sixth mass extinction? A view from the world of amphibians. Proceedings of the National Academy of Sciences of the USA 105:11466–11473. https://doi.org/10.1073/pnas.0801921105

    Article  PubMed  Google Scholar 

  60. Wells KD (2007) The Ecology and Behavior of Amphibians, Chicago, IL: The University of Chicago Press

    Book  Google Scholar 

  61. Wilson AC (2016) Distribution of cane toads (Rhinella marina) in Florida and their status in natural areas (Master’s Thesis). Retrieved from https://ufdc.ufl.edu/ufetd

  62. Yager DD (1992) Underwater acoustic communication in the African pipid frog Xenopus borealis. Bioacoustics 4:1–24. https://doi.org/10.1080/09524622.1992.9753201

    Article  Google Scholar 

  63. Yasumiba K, Alford RA, Schwarzkopf L (2015) Why do male and female cane toads, Rhinella marina, respond differently to advertisement calls? Animal Behaviour 109:141–147. https://doi.org/10.1016/j.anbehav.2015.08.015

    Article  Google Scholar 

  64. Yasumiba K, Alford RA, Schwarzkopf L (2016) Seasonal reproductive cycles of cane toads and their implications for control. Herpetologica 72(4):288–292. https://doi.org/10.1655/herpetologica-d-15-00048.1

    Article  Google Scholar 

  65. Zug GR, Zug PB (1979) The marine toad, Bufo marinus: a natural history resumé of native populations. Smithsonian Contributions to Zoology 1979(284):1–58. https://doi.org/10.5479/si.00810282.284

    Article  Google Scholar 

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Acknowledgements

We are grateful to N. Anderson, J. Manzano Alvarez, J. Peniston, B. Leavell, and H. Legett for collecting toads; G. Darnell, J. Lam, and E. Terry for assisting with dissections and measurements; and R. Knepp, J. Rusk, and C. Garcia Botero for analyzing photographic data. We are also thankful to K. McCoy, J. Lucas, and S. Johnson for providing valuable advice throughout the project as well as D. Carrillo and G. Nuessly for providing logistical support in Florida. Finally, we thank B. Muller, E. Bledsoe, and E. Khazan for providing comments that improved this manuscript, as well as J. Peniston, for designing figures.

Funding

This work was supported by funds from the Department of Biological Sciences at Purdue University to XEB and to SZ (Lindsey Graduate Fellowship) and by a student research grant from the Animal Behavior Society to SZ. SZ was supported by the Purdue Doctoral Fellowship from the Purdue University Graduate School. XEB was supported by the National Science Foundation (IOS #1433990).

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Correspondence to Sara Zlotnik.

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Experiments were approved by the Purdue Animal Care and Use Committee (Protocol #1405001073). All applicable institutional and/or national guidelines for the care and use of animals were followed.

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Zlotnik, S., Gridi-Papp, M. & Bernal, X.E. Laryngeal Demasculinization in Wild Cane Toads Varies with Land Use. EcoHealth 16, 682–693 (2019). https://doi.org/10.1007/s10393-019-01447-x

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Keywords

  • Anuran amphibian
  • Mating signal
  • Endocrine disruptor
  • Ecophysiology
  • Vocal cords
  • Rhinella marina (Bufo marinus)