Fig. 5.

Progression of infection in Galleria mellonella larva tissues after injection with Fno. Visualisation of G. mellonella larva tissues during 96 h after injection of Fno STIR-GUS-F2f7 in 10 μL phosphate-buffered saline at 1 × 10⁹ CFU/mL and incubation at 28 °C. Tissues were stained by haematoxylin and eosin (a, b, f, k, n, o), Gram Twort (c, d, g, h, i, l) or immunohistochemistry (IHC) with anti-Fnn primary antibodies that cross-react with Fno (e, j, m, p) in unmanipulated control larvae at 0 h (a-e) or larvae injected with Fno and sampled at 48 h (f-j), 72 h (k-h) and 96 h (n-p). Control larvae at 0 h showed scattered haemocytes in and around the fat body (a-c), subcuticular area (d) and in clusters surrounding the gastrointestinal tract (b); Fno was not detected by IHC (e). At 48 h, larvae injected with Fno showed infiltration of haemocytes into the fat body, eosinophilic fluid in the coelomic cavity (f) and enlarged haemocytes containing bacteria (g, h); melanised haemocytes were also observed (h). Clusters of haemocytes formed nodules, often surrounded by flattened cells (i), and Fno was detectable by IHC (j). At 72 h, large nodules had formed (k), and enlarged and melanised haemocytes were observed (l); great abundances of Fno cells were detected by IHC (m, p). At 96 h, large and increasingly melanised nodules were observed, while haemocytes at the periphery were flat in appearance (n); there was evidence for the recruitment of new, round haemocytes (n). Large protein lakes and severe tissue necrosis were observed (o) and great abundances of Fno cells were detected by IHC (p). Ct, cuticle; FB, fat body; GI, gastrointestinal tract; MF, muscle fibres; Me, melanin; Ne, necrosis; PL, protein lake; T, trachea. Scale bars: a, i = 20 μm; b, k, o = 100 μm; c, d, g, h, l = 10 μm; e, f, j, n, p = 50 μm