Germ Tube Mediated Invasion of Batrachochytrium dendrobatidis in Amphibian Skin Is Host Dependent

Abstract

Batrachochytrium dendrobatidis ( Bd ) is the causative agent of chytridiomycosis, a fungal skin disease in amphibians and driver of worldwide amphibian declines. We focussed on the early stages of infection by Bd in 3 amphibian species with a differential susceptibility to chytridiomycosis. Skin explants of Alytes muletensis , Litoria caerulea and Xenopus leavis were exposed to Bd in an Ussing chamber for 3 to 5 days. Early interactions of Bd with amphibian skin were observed using light microscopy and transmission electron microscopy. To validate the observations in vitro , comparison was made with skin from experimentally infected frogs. Additional in vitro experiments were performed to elucidate the process of intracellular colonization in L. caerulea . Early interactions of Bd with amphibian skin are: attachment of zoospores to host skin, zoospore germination, germ tube development, penetration into skin cells, invasive growth in the host skin, resulting in the loss of host cell cytoplasm. Inoculation of A. muletensis and L. caerulea skin was followed within 24 h by endobiotic development, with sporangia located intracellularly in the skin. Evidence is provided of how intracellular colonization is established and how colonization by Bd proceeds to deeper skin layers. Older thalli develop rhizoid-like structures that spread to deeper skin layers, form a swelling inside the host cell to finally give rise to a new thallus. In X. laevis , interaction of Bd with skin was limited to an epibiotic state, with sporangia developing upon the skin. Only the superficial epidermis was affected. Epidermal cells seemed to be used as a nutrient source without development of intracellular thalli. The in vitro data agreed with the results obtained after experimental infection of the studied frog species. These data suggest that the colonization strategy of B. dendrobatidis is host dependent, with the extent of colonization most likely determined by inherent characteristics of the host epidermis.

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

“…A germination tube then forms that penetrates cell membranes of the host, usually through more than one cell layer (Greenspan et al 2012;Van Rooij et al 2012). It is through this germ tube that cellular contents of the zoospore body are transferred to the host cell, where a zoosporangium then develops .…”

“…Zoosporangia are cleaved into daughter zoospores, and these then leave the zoosporangium through one or more 5 discharge papilla that are directed toward either the external body surface or intercellular spaces within the skin of the host ; Figure 1.3). However, in some hosts such as the tolerant species Xenopus laevis, growth of Bd appears to remain epibiotic, and does not penetrate past the stratum corneum, which is routinely shed (Van Rooij et al 2012). (A) A zoospore encysts on the epidermal surface, and then produces a germ tube that penetrates into the stratum corneum, or through one or more cell layers.…”

“…There is little definitive evidence of species demonstrating widespread resistance to chytridiomycosis, but some species appear to demonstrate greater resistance than most, including the notorious invader Lithobates catesbeianus (Gervasi et al 2013;Eskew et al 2015), and threatened species in the genus Leiopelma (Bishop et al 2009;), amongst others (Woodhams et al 2007a;Gahl et al 2012;Searle et al 2011b). Furthermore, additional species are thought to be largely tolerant to infection, most notably the common laboratory species Xenopus laevis from Africa (Ramsey et al 2010;Van Rooij et al 2012), and…”

“…However, if sloughing does not occur soon after exposure, encysted Bd zoospores may have more time to produce germination tubes to penetrate into deeper epidermal cell layers, beneath the stratum corneum (Greenspan et al 2012;Van Rooij et al 2012). …”

“…For example, in skin explants of the Australian green tree frog (Litoria caerulea), Bd was able to penetrate the outer skin layers within hours of encysting on the skin surface, resulting in exclusively intracellular growth (Van Rooij et al 2012). However, in skin explants of the African clawed frog (Xenopus laevis), a species tolerant of Bd infection, Bd infection remained epibiotic, and did not penetrate into deeper cell layers (Van Rooij et al 2012).…”