Feeding Drosophila highly radioresistant fungi improves survival and gut morphology following acute gamma radiation exposure

Abstract

Diverse fungi have been historically vital reservoirs of drug discovery, providing life-saving pharmaceuticals. Many species of fungi, yeasts in particular, are highly resistant to radiation, with their cellular contents potentially conferring dietary radioresistance. We developed a Drosophila model to test whether feeding two highly radioresistant fungi, Aureobasidium pullulans and Rhodotorula taiwanensis, could improve fly lifespan and gut morphology after acute irradiation. We constructed a dosimetry curve for the lifespan response of males and females to irradiation and found dose-dependent and sex-specific effects on lifespan. We also determined that the sex-specific response to irradiation correlated with nuclear morphology defects in the gut, with the more radiosensitive males displaying increased midgut cellular holes and aberrant nuclear morphology. To determine if feeding Aureobasidium pullulans and Rhodotorula taiwanensis before irradiation could improve survival and gut morphology, we first exclusively fed males and females each fungus and observed that they tolerated the diet well. Using these methods, we found that only two days of prefeeding Aureobasidium pullulans increased male lifespan, but not female, after irradiation, and improved nuclear morphology in the gut. However, dietary Rhodotorula taiwanensis was not protective. Overall, this study identified a highly radioresistant dietary fungus, Aureobasidium pullulans, as effective for extending male Drosophila lifespan and improving gut morphology following irradiation. Since the gut is particularly sensitive to the effects of irradiation, this fungus indicates a potential therapeutic for patients undergoing radiotherapy. Furthermore, this method could identify additional radioresistant fungi that protect the gut from radiation injury.

Keywords: Drosophila; fungi; gut; irradiation; lifespan; radioresistant.

Fig. 1:. Dosimetry and radiation sensitivity of Drosophila.

(A) Schematic of Drosophila irradiation sequence. (B) Lifespan of male and female flies acutely exposed to 0-1500 Gy gamma radiation. Radiation survival negatively correlates with dose. Males (dashed lines) were more sensitive than females (solid line) for all doses. (C) Graph showing a negative exponential relationship between the days to LD₅₀ for males and females and increasing IR dose, indicated by R² close to 1.0 for both sexes. (D) Lifespan of control (w¹¹¹⁸) and catalaseⁿ¹ (catⁿ¹) mutants after 1000 Gy acute irradiation. catⁿ¹ null flies are more sensitive to irradiation than control, and males are more sensitive than females for both genotypes. IR = ionizing radiation. Graphs were plotted using Microsoft Excel. Each point on the lifespan represents the average of triplicates of twenty flies that were irradiated simultaneously. Significance: (D) p = 0.0002 (***) for females and 0.000049 (****) for males.

Fig. 2:. Drosophila male guts are more sensitive to the effects of irradiation compared to females.

(A-D”) Dissected midguts from Day 4 adults exposed to IR (+IR) at day 2 and unexposed controls (−IR) labeled with phalloidin to label actin filaments and DAPI to label nuclei. The images of the R4 regions of midguts from each experimental condition were obtained using a confocal microscope with z-stacked for a layer of enterocytes. (A-A”) The enterocytes of a representative non-irradiated male control showing clear cellular barriers (A,A’) and good shape of nuclei (A,A”). (B-B”) The enterocytes of a representative male two days after irradiation showing abnormal nuclear shape (B, B”, arrowheads) and ambiguous cellular morphology due to disrupted cellular barriers (B,B’). (C-C”) The enterocytes of a representative female control and (D-D”) a representative female two days after irradiation showing normal nuclear morphology (C,C”,D,D”) compared to irradiated males (E). Irradiated females had disrupted cellular barriers between enterocytes (D,D’,F) similar to irradiated males (B,B’,F). (E-G) Each graph indicates the percentage of animals with guts showing abnormal nuclei (E), (disrupted cellular barriers (F), and holes in the actin filament layer in enterocytes (G). The quantification method is described in the Materials and Methods section. Each point on the graph represents the percentage of each replicate, and each bar represents the arithmetic mean of triplicates with a standard error of the mean (SEM). Data were analyzed statistically using a two-way ANOVA with multiple comparisons, followed by Tukey’s post hoc analysis. The following numbers of the midguts were assayed per experiment: male (−IR) (6, 7, 7); male (+IR) (5, 7, 4); female (−IR) (4, 8, 9); female (+IR) (5, 8, 9). Significance of multiple comparisons: (E) p = 0.0009 (***) for males and 0.0108 (*) for male (+IR) vs female (+IR); (F) p = 0.0016 (**) for males and 0.0030 (**) for females; (G) p = 0.0379 (*) for males. ns = not significant. (A,B,C,D) Green = phalloidin, blue = DAPI. (A’,B’,C’,D’) White = phalloidin. (A”,B”,C”,D”) White = DAPI. (A-G) −IR: non-irradiated, +IR: 1000 Gy irradiation. Scale bar: 10 μm in A for A-D”.

Fig. 3:. Rhodotorula taiwanensis and Aureobasidium pullulans are highly radioresistant fungi

(A) Characteristics of R. taiwanensis and A. pullulans. Both are highly radioresistant. (B) Agar plate showing streaked red R. taiwanensis. (C) Agar plate with a colony of white A. pullulans. (D) Lead shield used for irradiation experiments on an agar plate. Only the top half of the plate is exposed to irradiation. (E) Culture streak of A. pullulans protected (left) and unprotected (right) from exposure to irradiation. The right half of the culture is black due to melanin production following irradiation. (F, G) Micrographs of A. pullulans hyphae without (F, −IR) and with (G, +IR) irradiation. Melanin deposits (arrow) are visible in the hyphae. (H, I) Agar plate (H) and colony streak (I) of radiation-induced melanized A. pullulans allowed to grow without radiation. New fungal growth after irradiation is non-melanized and white (arrow heads). The colony shown in I is the same as the colony in E. Scale bar: 50 μm in G for F & G.

Fig. 4:. Drosophila tolerate dietary Aureobasidium pullulans and Rhodotorula taiwanensis

(A) Schematic of egg laying cups. Harvested R. taiwanensis and A. pullulans were placed on an agar plate for fly consumption (bottom). (B, C) Larva (B) and adult female fly (C) after feeding on R. taiwanensis. Red guts (arrows) indicate the animals ingested the fungi. (D, E) Larva (D) and adult female fly (E) after feeding on irradiated A. pullulans. Guts show the animals ingested the radiation-induced melanized fungi (arrows). (F) The percentage of larvae that pupated (solid lines) and eclosed (dashed lines) after being fed exclusively on alternative fungi. Each point on the graph represents the average of triplicates. Error bars represent standard deviation. (G) Lifespan analysis of males and females exclusively fed A. pullulans or R. taiwanensis. Agar only plates had no corn syrup (pink lines). Graphs were plotted using Microsoft Excel. Each point on the lifespan represents the average of triplicates of twenty flies that were fed fungi in parallel.

Fig. 5:. Feeding Aureobasidium pullulans improves lifespan in males

(A) Schematic showing the experimental timeline. Newly eclosed males and females are fed control yeast or experimental fungal paste for two days. The flies were subsequently placed in standard food vials and irradiated. Flies were transferred to fresh vials every 1-2 days until all died. (B) Lifespan of males fed non-irradiated A. pullulans (A.p.) (blue, −ɣ) and chronically irradiated A. pullulans (magenta, +ɣ) before exposure to 1000 Gy acute irradiation. (C) Lifespan of females fed non-irradiated A. pullulans (green, −ɣ) and chronically irradiated A. pullulans (orange, +ɣ) before exposure to 1000 Gy acute irradiation. Prophylactic dietary A. pullulans significantly extended the lifespan of males, but not females. (D) Lifespan of males fed nonirradiated R. taiwanensis (R.t.) (blue, −ɣ) and chronically irradiated R. taiwanensis (magenta, +ɣ) before exposure to 700 Gy acute irradiation. Feeding irradiated R. taiwanensis was detrimental to male lifespan. (E) Lifespan of females fed non-irradiated R.t. (green, −ɣ) and chronically irradiated R. taiwanensis (orange, +ɣ) before exposure to 700 Gy acute irradiation. Feeding nonirradiated and irradiated R. taiwanensis to females significantly decreased their lifespan. Graphs were plotted using Microsoft Excel. Each point on the lifespan represents the average of triplicates of twenty flies that were irradiated simultaneously. Error bars represent standard deviation. Statistical analysis was calculated using Online Application for Survival Analysis 2 (OASIS) and statistical significance was calculated using the Wilcoxon-Breslow-Gehan test. P values vs control are as follows: (B) p = 0.0331 (*) for A.p.(+ɣ), 0.0297 (*) for A.p.( −ɣ) ; (C) p=0.461 for A.p.(+ɣ), 1.0 for A.p.(−ɣ) (D) p= 0.0041 (*) for R.t.(+ɣ), 1.0 for R.t.(−ɣ) (E) p= 0.0002 (***) for R.t.(+ɣ), 0.0116 (*) for R.t.(−ɣ).

Fig. 6:. Feeding Aureobasidium pullulans attenuates IR-induced cellular damage in male guts

(A-D”) Dissected midguts from day 4 adult males exposed to IR at day 2 and control males labeled with phalloidin to label actin filaments and DAPI to label nuclei. The images of the R4 regions of midguts from each experimental condition were obtained using a confocal microscope with z-stacked for a layer of enterocytes. (A-A”) Representative gut dissected from a male control, showing clear cell barriers (A,A’) and normal nuclear shapes in enterocytes (A,A”). (B-B”) Representative gut dissected from a male two days after irradiation. Enterocytes had aberrant nuclear shape (B, B”, arrowheads, E) and altered cellular morphology (B,B’,F). Holes were formed within a layer of actin filament (B,B’, asterisk, G) by irradiation. (C-C”) Representative gut from a male fed A. pullulans (AP) had normal nuclear morphology (C,C”,E) in enterocytes while having a loss of cell barriers with some degree (C,C’,F). (D-D”) Representative gut from a male fed A. pullulans followed by irradiation had normal nuclear shape (D,D”,E) as well as loss of cellular barriers and irregular cell shape (D,D’,F). Many small holes appeared within actin layers (D,D’, number sign) but were not counted because they did not satisfy the criterion as described in Methods. Each graph indicates the percentage of animals having guts with abnormal nuclei (E), (F) disrupted cellular barriers (F), or holes in the actin filaments layer in enterocytes (G). Quantification method is described in the Materials and Methods section. Each point on the graph represents the percentage of each replicate, and each bar presents the arithmetic mean of triplicates with a standard error of the mean (SEM). Data were analyzed statistically using a two-way ANOVA with multiple comparisons, followed by Tukey’s post hoc analysis. The following numbers of the midguts were assayed per experiment: male control (11, 6, 7); male +IR (8, 4, 10); male fed A.p. (8, 6, 5); male fed A.p.−IR (7, 8, 7). P values of multiple comparisons are as follows: (E) p = 0.0026 (**) for (−/+IR) without A.p. and 0.0197 (*) for (−/+A.p.) with IR; (F) p < 0.0001 (****) for (−/+IR) without A.p., 0.0007 (***) for (−/+IR) with A.p., and 0.0014 (**) for (−/+A.p.) without IR; (G) p = 0.0225 (*) for (−/+IR) without A.p. ns = not significant. (A,B,C,D) Green = phalloidin, blue = DAPI. (A’,B’,C’,D’) White = phalloidin. (A”,B”,C”,D”) White = DAPI. (A-G) −IR: non-irradiated, +IR: 1000 Gy irradiation. (E,F,G) – A.p.: non-feeding A. pullulans, +Ap: feeding A. pullulans. Scale bar: 10 μm in A for A-D”.