Temperature-dependent growth of Geomyces destructans, the fungus that causes bat white-nose syndrome

Abstract

White-nose syndrome (WNS) is an emergent disease estimated to have killed over five million North American bats. Caused by the psychrophilic fungus Geomyces destructans, WNS specifically affects bats during hibernation. We describe temperature-dependent growth performance and morphology for six independent isolates of G. destructans from North America and Europe. Thermal performance curves for all isolates displayed an intermediate peak with rapid decline in performance above the peak. Optimal temperatures for growth were between 12.5 and 15.8°C, and the upper critical temperature for growth was between 19.0 and 19.8°C. Growth rates varied across isolates, irrespective of geographic origin, and above 12°C all isolates displayed atypical morphology that may have implications for proliferation of the fungus. This study demonstrates that small variations in temperature, consistent with those inherent of bat hibernacula, affect growth performance and physiology of G. destructans, which may influence temperature-dependent progression and severity of WNS in wild bats.

Figure 1. Weekly growth curves for two isolates of Geomyces destructans.

In an initial experiment, two isolates of G. destructans (one from New York and one from Germany) exhibited differences in growth performance but had similar thermal optima and upper critical temperatures for growth. T_opt and upper critical temperatures (CL_u) for growth at week 5 are marked on the graphs with arrows. For this figure, each curve is represented using a Brière2 function, although in some cases, other functions were equally parsimonious (Table S1). T_opt and CL_u in this figure represent the values specific to the Brière2 function shown in the graph; therefore T_opt does not match the weighted averages presented in Table 1. The isolates were grown on Sabouraud dextrose agar. Twenty-one replicate colonies of each were incubated across a range of nine temperatures from 0.8 to 21.4°C. The area of each expanding colony was measured weekly, for five weeks, and a growth curve was fit to each weekly dataset.

Figure 2. Five-week growth curves for four isolates of Geomyces destructans.

In a follow-up experiment, differences in growth performance were confirmed among four additional isolates of G. destructans, two from North America and two from Europe. A consistent intercontinental trend in growth performance was not observed among the isolates. The isolates were grown on Sabouraud dextrose agar. Twenty-one replicate colonies of each isolate (Pennsylvania, Virginia, Hungary, and Switzerland) were incubated across a range of five temperatures from 1.9 to 17.7°C. The area of each expanding colony was measured after five weeks, and a growth curve was fit to each dataset. Each curve is represented using the best-fit function. For comparison to the weekly growth curve analysis (Fig. 1), 21 replicate colonies each of the isolates from New York and Germany were incubated with the other isolates at 6.7, 12.2, and 17.7°C. The area of each expanding colony was measured after five weeks, and descriptive data (mean ± SD) are represented. Isolates from New York and Germany grew proportionally faster at each of the temperatures used for this analysis than in the initial weekly analysis. Although the curve shapes for both analyses were consistent, the two analyses cannot be directly compared.

Figure 3. Morphology of Geomyces destructans varies with incubation temperature.

(a) A characteristically branched conidiophore following growth at approximately 7°C. (b) Curved conidia typical of those produced following incubation at approximately 7°C. (c) Hyphae were thickened, fragmented into arthrospores (arrows), and produced chlamydospore-like structures (arrowhead) following incubation at approximately 12°C. (d) Conidia were primarily pyriform to globoid in shape and frequently formed short chains (arrow) following incubation at approximately 12°C. (e) At elevated temperatures (above 15°C), thickened, deformed hyphae showed evidence of degeneration, and hyphal tips exhibited branched antler-like morphology (arrow). Chlamydospore-like structures were also common (arrowhead). (f) Thick irregular hyphal fragments were produced by colonies grown at approximately 18°C; conidia were not observed. Scale bars, 10 µm.