Innate immune defenses of amphibian skin: antimicrobial peptides and more

Abstract

Chytridiomycosis is an emerging disease of amphibians with global impact (Skerratt et al., 2007). Innate immune defenses may contribute to the ability of some species to coexist with endemic Batrachochytrium dendrobatidis (Bd) (Retallick, McCallum & Speare, 2004; Woodhams et al., 2007b). We appreciate the thoughtful commentaries by Fisher (2007), Garner (2007) and Kurtz & Scharsack (2007) to the paper ‘Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses’ (Woodhams et al., 2007a). The commentators endorsed the ecological immunology approach of the work and its utility for informing conservation. They included excellent suggestions for future studies including the following: (1) Examining population, family and individual level variation in amphibian immune defenses. (2) Testing for mucosal antibodies, immune memory and induction of innate defenses in addition to constitutive innate defenses. (3) Determining how behavioral differences among species affect the rate of encountering the pathogen and response to encounter. (4) Understanding how environmental conditions affect innate immunity. The commentators pointed out that immunity can vary among lineages of frogs (Pearman & Garner, 2005) and stickleback fish (Rauch, Kalbe & Reusch, 2006), suggesting that some of the among-species differences we detected might be due to differences among lineages within species. This is true, however, as the work cited by the commentators illustrates, fully exploring variation in disease susceptibility within even a single species requires very large, complex experiments. For our initial exploration of variation among species, we chose to use single sibships to minimize withinspecies variation, maximizing the power of our comparisons among species. This also minimized the probability of chance differences in peptide expression between infected and control frogs. This was advantageous because we could collect peptides only from uninfected control frogs, because depletion caused by norepinephrine induction could have affected the survival of infected frogs. We agree that much remains to be investigated in terms of variation in innate immunity among sibships and populations; however, it seems likely that variation of skin peptide defenses among species is greater than within-species variation. For example, Apponyi et al. (2004) found substantial variation on a large geographic scale in the composition of skin peptides profiled within two species of Australian treefrogs, and also showed that most species produced profiles containing distinctive families of peptides, and that on smaller geographical scales profiles did not differ among individuals within species. Our own studies of peptide variation among individuals of two populations of Rana muscosa in the Sierra Nevada Mountains of California suggest very limited variation in skin peptides (Woodhams et al., 2007b). This suggests that sibship-level differences in skin peptide defenses are not likely to have affected the results of this study in which four species from the same region were compared. Some antimicrobial peptides are constitutive and others may be induced in response to infection (Boman, Nilsonn & Rasmuson, 1972; Zasloff, 2002; Cunliffe & Mahida, 2004; Izadpanah & Gallo, 2005). Here, peptide induction has two meanings: transcription and storage in glands or secretion from glands onto the skin. We did not test for induction of skin peptides in response to Bd exposure in this study. However, the constitutive peptide defenses may be a good measure of immune condition before Bd exposure. Any induction of new skin peptides that may occur in response to Bd infection may not be effective because chytridiomycosis leads to mortality of susceptible species between 18 and 48 days post-infection (Berger et al., 2004; Woodhams et al., 2007a), and production of peptides in granular glands after discharge can take as long as 6 to 421 days (Flucher et al., 1986; Giovannini et al., 1987; Rollins-Smith & Conlon, 2005). Of course the quantity of peptides released from the granular glands after administration of norepinephrine is