Larval Environment Alters Amphibian Immune Defenses Differentially across Life Stages and Populations

RESEARCH ARTICLE Larval Environment Alters Amphibian Immune Defenses Differentially across Life Stages and Populations Katherine L. Krynak1☯*, David J. Burke2☯, Michael F. Benard1☯ 1 Department of Biology, Case Western Reserve University, Cleveland, Ohio, United States of America, 2 Research Department, The Holden Arboretum, Kirtland, Ohio, United States of America ☯ These authors contributed equally to this work. * Abstract OPEN ACCESS Citation: Krynak KL, Burke DJ, Benard MF (2015) Larval Environment Alters Amphibian Immune Defenses Differentially across Life Stages and Populations. PLoS ONE 10(6): e0130383. doi:10.1371/journal.pone.0130383 Editor: Vincent Laudet, Ecole Normale Superieure de Lyon, FRANCE Received: January 28, 2015 Accepted: May 20, 2015 Published: June 24, 2015 Copyright: © 2015 Krynak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Data obtained for this study is archived in DataDryad (http://dx.doi.org/10. 5061/dryad.1872r/1). Funding: This material is based upon work supported by the National Science Foundation Graduate Research Fellowship, under Grant No. DGE-0951783, to KLK, fieldwork was supported by Case Western Reserve University to MFB, and laboratory work was supported by The Holden Arboretum Trust to DJB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Recent global declines, extirpations and extinctions of wildlife caused by newly emergent diseases highlight the need to improve our knowledge of common environmental factors that affect the strength of immune defense traits. To achieve this goal, we examined the influence of acidification and shading of the larval environment on amphibian skin-associated innate immune defense traits, pre and post-metamorphosis, across two populations of American Bullfrogs (Rana catesbeiana), a species known for its wide-ranging environmental tolerance and introduced global distribution. We assessed treatment effects on 1) skinassociated microbial communities and 2) post-metamorphic antimicrobial peptide (AMP) production and 3) AMP bioactivity against the fungal pathogen Batrachochytrium dendrobatidis (Bd). While habitat acidification did not affect survival, time to metamorphosis or juvenile mass, we found that a change in average pH from 7 to 6 caused a significant shift in the larval skin microbial community, an effect which disappeared after metamorphosis. Additionally, we found shifts in skin-associated microbial communities across life stages suggesting they are affected by the physiological or ecological changes associated with amphibian metamorphosis. Moreover, we found that post-metamorphic AMP production and bioactivity were significantly affected by the interactions between pH and shade treatments and interactive effects differed across populations. In contrast, there were no significant interactions between treatments on post-metamorphic microbial community structure suggesting that variation in AMPs did not affect microbial community structure within our study. Our findings indicate that commonly encountered variation in the larval environment (i.e. pond pH and degree of shading) can have both immediate and long-term effects on the amphibian innate immune defense traits. Our work suggests that the susceptibility of amphibians to emerging diseases could be related to variability in the larval environment and calls for research into the relative influence of potentially less benign anthropogenic environmental changes on innate immune defense traits. PLOS ONE | DOI:10.1371/journal.pone.0130383 June 24, 2015 1 / 22 Larval Environment Alters Amphibian Immune Defenses Competing Interests: The authors have declared that no competing interests exist. Introduction Although it is well accepted that phenotypes vary between populations and are influenced by environmental conditions, there is increasing interest in the effects of environmental change on traits that affect resistance to newly emerging pathogens [1,2,3]. There is a large body of evidence indicating that environmental change, including human induced changes such as increasing temperatures, deforestation, and acidification, can alter an organism’s growth, development and survival [4,5, 6,7,8,9]. However, relatively few studies have experimentally examined the effects of environmental change on immune defense traits, which may greatly affect individual health [10,11,12, 13]. Given the rapid global spread of infectious diseases, a better understanding of the environmental factors that govern the expression of immune defense traits, and how this response to environmental change varies between populations and across life-stages, is increasingly needed. Amphibians are an excellent study group for examining the role of the environment in regulating immune defense traits. Many of the diseases associated with amphibian declines either enter the amphibian through the dermal tissue (i.e. skin), or directly affect the dermal tissue (e.g. chytridiomycosis caused by the fungus Batrachochytrium dendrobatidis) [14,15,16]. Many amphibians possess two innate traits which resist pathogen infection of the skin. First, amphibian adults and larvae harbor diverse microbial communities on their skin [17]. Some amphibian skin-associated microbial species produce metabolites that suppress and eliminate some amphibian diseases [18,19,20,21,22,23]. Second, antimicrobial peptides (AMPs) produced by the granular glands of amphibian skin provide an effective defense against a variety of pathogens by disrupting pathogen cell and viral membranes [24,25,26]. How changes in the environment affect skin-associated microbial communities and AMPs has not been widely examined [27]. The small number of studies which have examined the effect of the environment on amphibian innate immune defense traits are largely correlative [17,28,29] and few have applied experimental manipulations to examine how environmental factors affect these traits [11,12,30,31,32]. Experimental studies provide conflicting evidence on the degree to which the environment may influence amphibian immune defense traits; some studies have found that immune defenses are not altered by the environment [11,12,17] whereas other studies have found environmental effects on immune defenses [30,32]. Even commonly encountered variations to amphibian habitat may alter immune defense traits as has been routinely found in studies examining traits associated with growth and development [33,34]. Additionally, commonly encountered variations in the environment may affect immune defense traits across life stages. Several studies have found alterations to larval habitat including changes in canopy cover, pond ephemerality, po (...truncated)