Interactions between amphibian skin sloughing and a cutaneous fungal disease: infection progression, immune defence, and phylogenetic patterns

Abstract

Worldwide, there has been an unprecedented rise in emerging infectious diseases of wildlife, and this has contributed to a widespread biodiversity crisis. Amphibian populations, in particular, are threatened by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which in post-metamorphic animals only infects the skin, and causes the potentially lethal disease chytridiomycosis. Amphibians regularly slough their skin, and in doing so remove many skin- associated microbes. Thus, skin sloughing may play an important role in the pathogenesis of chytridiomycosis. To investigate this association, the influence of Bd infection on amphibian skin sloughing, and the role of sloughing in regulating infection, was examined. Furthermore, to better understand the variation in skin sloughing rates across species and ecological groups, and make inferences about the role of this process in susceptibility to this fungal disease, amphibian skin structure and function was investigated within a phylogenetic context. To determine the relationship between skin sloughing and disease progression (chapter 2), adult green tree frogs (Litoria caerulea) were exposed to an Australian Bd strain, and sloughing rates and infection load were monitored on a naturalistic cycling temperature regime (15 - 23 C). Sloughing rates were determined by filming frogs and infection intensity was monitored before and after sloughing with conventional swabbing and quantitative PCR. Sloughing rate was found to increase with Bd infection load in infected frogs, but sloughing itself did not affect Bd load on the ventral skin surface. Although a faster sloughing rate might be considered advantageous for Bd- infected animals, it does not appear to curb the progression of disease in susceptible species. In fact, sloughing may actually contribute to the loss of physiological homeostasis seen in terminally ill frogs by further inhibiting water and electrolyte transport across the skin. In some species less susceptible to chytridiomycosis, it has been demonstrated that Bd growth remains epibiotic, without penetrating the underlying epidermal layers. Therefore, sloughing may more effectively remove Bd zoospores in less susceptible species (chapter 3). To test this hypothesis, five Australian frog species, Lit. caerulea, Platyplectrum ornatum, Lechriodus fletcheri, Limnodynastes peronii, and Lim. tasmaniensis, were exposed to an Australian Bd strain, and their sloughing rates and infection loads monitored over time. Utilising an improved methodology to remove any artefacts from the swabbing itself, sloughing was found to reduce Bd load on the ventral skin surface, in all five species, despite wide ranging variation in susceptibility to Bd infection and subsequent disease. In less susceptible species, sloughing reduced Bd load up to 100%, leading to infection clearance. However, the drop in Bd load was only temporary in susceptible species, potentially due to the invasive growth of Bd in skin layers underlying the stratum corneum in these species. If less susceptible species are able to clear themselves of Bd infection via the routine process of skin shedding, amphibian sloughing may be a more important immune defence than previously thought. This work has implications for understanding the pattern of Bd growth on individual hosts, as well as population-level dynamics. Finally, the relationship between susceptibility to chytridiomycosis and skin structure and function between species was investigated within a phylogenetic context (chapter 4). The sloughing rates of 21 frog species from around the globe were measured, including Australia (9), Central and South America (11), and Southeast Asia (1). In addition to measuring sloughing rates, epidermal thickness and the number of replacement layers in preserved specimen of seventeen of these species were also measured. Utilising a phylogenetic linear mixed model framework, the association of these skin turnover traits with the evidence for Bd-driven declines was assessed, based on information from the IUCN Red List, published papers, grey literature, and personal communications. It was determined that sloughing rate demonstrates high phylogenetic signal, but was not associated with the evidence of Bd-driven declines, or other skin characteristics, within this subset of species. This is the first comparison of sloughing rate across a wide range of amphibian species, and creates the first database of amphibian sloughing behaviour. Given the strong phylogenetic signal observed in sloughing rate, approximate sloughing rates of related species may be predicted based on phylogenetic position, and may help to explain differences in the severity of infection in genera with relatively slow skin turnover rates (e.g. Atelopus). A clear understanding of epidermal turnover in amphibian genera particularly affected by Bd may help focus conservation mitigation efforts. Despite the restriction of Bd to the post-metamorphic amphibian epidermis, the role of skin sloughing as an immune defence mechanism has so far been overlooked. This work investigating the physiology of amphibian skin and the host-pathogen relationship at the site of pathogen colonisation brings us closer to understanding the factors driving observed variation in intra- and interspecific susceptibility across a wide range of amphibian hosts. This understanding can help improve species-specific predictions of host extinction risk in natural populations.