Figure 4
Zygosaccharomyces sp. lipid droplets and S. depilis pupation experiments. (a) Fluorescence microscopy of Zygosaccharomyces sp. fixed cells stained by Nile Red to show the presence of cytoplasmic lipid droplets under laboratory conditions and (b) natural conditions (cells of the fungus collected directly from brood cells of S. depilis). (c) Different stages of larval development: (i) 1–2 days-old egg, (ii) 3–4 day-old egg, (iii) 1 day-old larva, (iv) 3–4 day-old larvae (Zygosaccharomyces sp. growth observed), (v) 6–10 day-old larva, (vi) 15–18 day-old pre-pupa, (vii) 20–25 day-old pupa, (viii) 30–34 day-old pupa, (ix) 35–40 day-old emerging bee. (d) Percentage of larvae that completed metamorphosis in vitro without microorganism inoculation (first bar), with fungus collected directly from brood cells (second bar) and with isolated Zygosaccharomyces sp. SDBC30G1, cultivated in laboratory (third bar), in three different experiments (N = 48 per treatment, Cochran-Mantel-Haenszel, P < 0.0001 compared with uninoculated control). (e) Percentage of larvae that completed metamorphosis in vitro without fungal or sterol inoculation (first bar), with fungus collected directly from brood cells (second bar) and with ergosterol at 2.5 µM added in the larval food (third bar), in three different experiments (N = 48 per treatment, Cochran-Mantel-Haenszel, P < 0.0001 compared with untreated control). (f) Chemical structures of ergosterol, cholesterol, 24-epi-makisterone A and 20-hydroxyecdysone.
