Susceptibilityto Chytridiomycosis

Considerable variation in susceptibility to chytridiomycosis has been observed between hosts at the individual, population and species levels and is attributable to both extrinsic and intrinsic host factors including immunological defences (Blaustein et al.

14.6.1 ExtrinsicHostFactors

At the broad scale, population declines due to Bd appear strongly linked to species' distribution and habitat use. Declines have been particularly severe among tropical high-altitude, stream-associated anurans, although in many cases, susceptibility and population declines are likely associated with environmental suitability for the fungus rather than innate variations between species (Lips et al. 2006; Fisher et al. 2009). Even among syntopic species, however, habitat use may differ which may explain some degree of variation in perceived susceptibility to chytridiomycosis (Rowley and Alford 2007).

In Central and South America, the Atelopus genus of neotropical toads appears to have been most severely affected (La Marca et al. 2005). In Australia, diverse species of Myobatrachid and Hylid frogs have become extinct or are critically endangered (Skerratt et al. 2016). Globally, Bd-associated declines and extinctions have been recorded in numerous other amphibian taxa, particularly other anurans (Fisher et al. 2009). Many salamanders may also have been significantly affected (Rovito et al. 2009; Cheng et al. 2011). There are several host species that are predominantly resistant to or tolerant of infection and may serve as reservoirs or carriers of infection. These species may have been responsible for the widespread dissemination of Bd and include, for example, Pseudacris regilla, L. catesbeianus, L. pipiens, X. laevis, Eleutherodactylus coqui, Litoria Iesueurii complex, L. ewingii and Crinia signifera (Retallick et al.

Behaviour that increases exposure to the pathogen may constitute an important extrinsic host factor contributing to variation in susceptibility. This may be through types and degrees of host contact with conspecifics, contaminated water and envi­ronmental substrates containing Bd (Rowley and Alford 2007). Importantly, how­ever, other general movement patterns, including thermoregulatory behaviours such as basking, and the use of retreat sites may also play a role by altering existing infection intensities or pathogen growth rates due to the optimal thermal range of the fungus (Rowley et al. 2007; Richards-Zawacki 2010; Puschendorf et al. 2011; Daskin et al. 2011). Behaviour may differ particularly between life stages due to aquatic and terrestrial or arboreal habitats as well as between male and female adults due to their differing seasonal utilisation of the environment (Duellman and Trueb 1994). Other factors may include the density of individuals within the population (higher densities may increase environmental zoospore load; Hudson and Dobson 1998), the age structure of the population (tadpoles may act as reservoir hosts, or dispersal patterns and habitat use differ between stages; Rachowicz and Vredenburg 2004) and the presence of and interactions with any syntopic species and vectors (Reeder et al. 2012; Rivas 1964).

14.6.2 IntrinsicHostFactors

Intrinsic host factors associated with signalment (life stage, age and body size) have been found to differentiate susceptibility to chytridiomycosis between individuals in laboratory experiments and field observations. Infections are limited to the keratinized mouthparts of larval anurans and are typically not fatal during this stage (Berger et al. 1998; Pessier et al. 1999; Fellers et al. 2001; Rachowicz and Vredenburg 2004), although infection prevalence and intensity has been found to increase with larval development (Smith et al.

Controlling for the factors described above, experimental evidence suggests the importance of both genetic and phenotypic immunologic mechanisms in differentiating susceptibility between individuals, clutches, populations and species (Rosenblum et al. 2012; Grogan 2014). These variations in response may be associated with inherent or evolved differences in innate immunity (Savage and Zamudio 2011; Bataille et al. 2015), previous exposure history and the development of adaptive immunity (Cashins et al. 2013; McMahon et al. 2014) or phenotypic differences in pathogenesis and differing functional expression of immune responses. While predisposing immunosuppression is not necessary for epidemics to occur, apparent immunosuppression has been observed in Bd-infected individuals (detected via skin histopathology, and corroborated via gene expression and in vitro immune studies (Berger et al.

14.6.2.1 InnateImmuneResponse

Many components of the innate immune response to Bd infection differ between experimental groups and may be associated with differential host susceptibility. The secreted mucus and epidermal layer of amphibian skin provide a constitutive physical and chemical defence barrier against pathogen invasion and may include variable expression of lysozyme (Rollins-Smith 2009), mucosal antibodies, induc­ible AMPs (such as defensins, cathelicidins and histatins; Ramsey et al. 2010; Pask et al. 2013) as well as commensal symbiotic bacterial communities and their antimicrobial metabolites (Lauer et al. 2007; Harris et al. 2009a, b).

14.6.2.2 Antimicrobial Peptides

AMPs are small (12-50 amino acid residues), cationic and hydrophobic peptides produced in the dermal granular glands. Most of our current knowledge concerning amphibian AMPs stems from studies on Anura (Van Rooij et al. 2015). To date, approximately 40 anuran AMPs inhibiting Bd have been characterized. Both purified and natural mixtures of these AMPs effectively inhibit in vitro growth of both Bd zoospores and sporangia (Woodhams et al. 2007; Rollins-Smith 2009; Ramsey et al. 2010). However, it is not clear to which extent these peptides provide protection against chytridiomycosis in vivo (Van Rooij et al. 2015). Species with peptides active in vitro such as the mountain yellow-legged frog (Rana muscosa) may still be very susceptible to Bd (Rollins-Smith et al. 2006). Moreover, the efficacy of skin peptide defences may vary at species and population level (Tennessen et al. 2009).

14.6.2.3 Bacteria and Their Antifungal Metabolites

Several secondary metabolites secreted by symbiotic bacteria present on amphibian skin have been shown to inhibit Bd growth in vitro and in vivo (Van Rooij et al.

2015). In the bacteria Janthinobacterium lividum, Lysobacter gummosus and Pseu­domonas fluorescens, these metabolites are 2,4-diacetylphloroglucinol (2,4-DAPG), indol-3-carboxaldehyde (I3C) and violacein (Brucker et al. 2008; Myers et al. 2012; Lam et al. 2010; Harris et al. 2009a, b). In addition, the metabolites 2,4-DAPG and I3C seem to exert a repellent action on Bd zoospores (Lam et al. 2011). Besides, coculture of skin bacterial isolates can lead to secretion of new, more potent metabolites than when grown in monoculture. As such, the inhibitory metabolite tryptophol was found to emerge from coculturing an unknown Bacillus skin bacte­rium and Chitinophaga arvensicola (Loudon et al. 2014). Myers et al. (2012) showed that these metabolites work synergistically with AMPs to inhibit growth of Bd, at lower minimal inhibitory concentrations (MIC) than necessary for inhibi­tion by either metabolites or AMPs. As for AMPs, variation in infection susceptibil­ity among populations is thought to result in part from differences in skin bacterial communities (Van Rooij et al. 2015). By comparing bacterial communities on the skin of a declining R. muscosa population and a population coexisting with Bd, researchers found a significantly higher number of individuals with culturable bacterial species displaying antifungal properties in coexisting populations than in those at decline. Alteration of this microbial community composition, for example, by environmental factors, can considerably increase susceptibility to disease (Lam et al. 2010).

14.6.2.4 Other Inducible Pathways of the Innate Immune System

Zoospores that survive to invade beyond the skin mucus layer induce host innate immune responses via antigens that are either secreted or expressed on the pathogen cell surface or released through lysosomal degradation within host cells. These antigens often contain epitopes of widely recognized pathogen-associated molecular patterns (PAMPs) that are common to many different microorganisms.

14.6.2.5 Adaptive Immune Response

There is currently little evidence to suggest the effective activation of the adaptive immune response to chytridiomycosis in terms of a systemic or localized lymphocyte response (Pessier et al. 1999; Berger et al. 2005; Peterson et al. 2013; Young et al. 2014; Nichols et al. 2001) that may differentiate susceptible from resistant individuals. Mapping of transcriptomic changes in immunologically important tissues (skin, liver, spleen, small intestine) from frogs with Chytridiomycosis demonstrated the absence of a robust adaptive immune response at various time points post exposure in various species (e.g. lymphocyte, immunoglobulin, MHC and classical comple­ment pathway genes; Ribas et al. 2009; Rosenblum et al. 2009; Ellison et al. 2014). Attempts to immunize frogs by subcutaneous or intraperitoneal injection of formalin (R. muscosa; Stice and Briggs 2010) or heat-killed Bd (Bufo boreas; Rollins-Smith 2009) failed to elicit a protective immune response. Only in X. laevis, Bd-specific IgM, IgX (mammalian IgA-like) and IgY (mammalian IgG-like) antibodies were found in skin mucus after injection with heat-killed zoospores (Ramsey et al. 2010). Although repeated exposure to Bd did not result in increased resistance in all experiments (Cashins et al. 2013), repeated cycles of exposure to Bd with subsequent temperature treatment of infection resulted in a marginally higher survival rate, reduction in the infection load (Ramsey et al. 2010; McMahon et al. 2014), which coincided with increased lymphocyte proliferation and abundance in the spleen. As such, some species of frogs are capable of acquiring at least some degree of immunity against Bd (McMahon et al. 2014), although in general, adaptive immune responses are suppressed by Bd.

14.6.3 Host Defences Against Bsal

Little is known about amphibian defences against Bsal. Unlike for anurans, infor­mation about the AMP arsenal in skin secretions of urodelans is scant (Van Rooij et al. 2015). To date only a single antimicrobial peptide (the defensin CFBD) has been described from Cynops fudingensis (Fuding fire belly newt) (Meng et al. 2013). Although the antimicrobial action of CFBD against both Batrachochytrium species has not (yet) been evaluated, it is suggested that AMPs may be involved in the pronounced anti-Bd activity of salamander skin secretions (Sheafor et al. 2008; Pasmans et al. 2013). Infection trials demonstrated that host responses vary dramati­cally, not only within but also between urodelan species (Martel et al. 2014). Interestingly, in some species that are likely Bsal reservoirs, a proportion of the exposed individuals do not develop lethal skin disease but are capable of self-cure after initial infection, in some cases with complete fungal elimination. This process can take several months and is most likely due to either increased innate defence mechanisms or the buildup of protective acquired immunity. Other urodelan species, however, which were shown to invariably develop lethal disease, do not seem to increase resistance against Bsal, even after five cycles of exposure and thermal treatment (unpublished results).

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