CantonS flies were exposed or mock-exposed (control) to E. muscae 'Berkeley' starting 3–5 hr after the light-dark transition and were subsequently sampled at 24, 48, 72 or 96 hr for histological analysis. For each time point, 4–6 individual, paraffin-embedded flies were sectioned at 8 µm, stained using Safranin O/Fast Green to identify fungal morphology and location and imaged at 20x magnification (Zeiss Axio Scan.Z1 slide scanner). Representative images from each time point are shown. Only male flies are shown here for ease of comparison. No differences in the progression of the infection were observed between males and females. An inset of the brain and the abdomen are shown for each sample. (A) Uninfected fly with major anatomical features labeled as follows: e - eye, b - brain, g - gut, t - testes, fb - fat body. (B) At 24 hr after exposure there is significant inflammation observed in the abdominal fat body (black arrowheads label a few clear examples); the nervous system is devoid of fungal cells. (C) At 48 hr after exposure E. muscae 'Berkeley' cells are present in the brain (white arrowheads) and/or ventral nerve cord (VNC) of all but one sample where E. muscae 'Berkeley' cells abut but have not yet entered brain. E. muscae 'Berkeley' cells are sparsely observed in the abdominal and/or thoracic hemolymph at 48 hr. The gut and testes are not invaded by fungus. (D) At 72 hr after exposure, E. muscae 'Berkeley' can be found throughout the body cavity and the amount of visible fat body has decreased. E. muscae 'Berkeley' titers have increased in the nervous system. (E) In a living fly at 96 hr after exposure (the first point at which a fly may be killed by E. muscae 'Berkeley' infection), fungus occupies virtually all available volume in the hemolymph. E. muscae 'Berkeley' titers have increased in the nervous system, gut and gonads remain uninvaded. (F) In an E. muscae 'Berkeley'-killed fly (cadaver) at 96 hr after exposure, only traces of host organs remain in the abdomen and the nervous system has been considerably degraded. No fat body cells are observed. E. muscae 'Berkeley' cells differentiate into conidiophores, cell-walled structures that will pierce through weak points of the cuticle to produce and launch infectious conidia. Black scale bars are 100 µm. All living animals shown are males; cadaver’s sex is undetermined (the gonads have been consumed by the fungus).