Fig 1.
Effect of D. cf. concentrica culture on L1–L2 larvae of C. elegans.
L1–L2 larvae of WT C. elegans were exposed for 48 h to three 50-mm diameter D. cf. concentrica culture plates. The viable nematodes passed through the 30-μm sieve and were counted. Ten replicates were used for each treatment with 300 nematodes per replicate. Graphs are representative of three independent experiments. Significant differences (as indicated by independent Student’s t-test) between control and Daldinia treatments are denoted by an asterisk (P ≤ 0.05).
Fig 2.
Effects of individual VOCs and SVM on WT C. elegans.
(A) Eggs, (B) L1, (C) L4 and (D) YA larval stages. Eggs or nematodes were exposed to 3-methyl-1-butanol (0.1 mL, 0.92 mmole), (±)-2-methyl-1-butanol (0.1 mL, 0.93 mmole), 4-heptanone (0.2 mL, 1.43 mmole), isoamyl acetate (0.1 mL, 0.68 mmole), or up to 0.5 mL of SVM. The viable nematodes were passed through sieves and counted. Data on the y-axis are presented as no. of eggs hatching (A) and as percent mortality relative to controls (mean ± SE) (B-D). Graphs are representative of three independent experiments. Pairwise comparison of the means was performed with analysis of variance followed by post-hoc Tukey–Kramer multiple comparison test. Different letters indicate significantly different values (P ≤ 0.05).
Fig 3.
Effects of 4-heptanone and SVM on C. elegans development.
Representative bright-field images of (A) eggs, (B) L1 and (C) L4 larvae on an E. coli lawn exposed to 4-heptanone (0.2 mL, 1.43 mmole) or SVM for 48 h. Scale bar = 100 μm.
Fig 4.
Comparison of the effects of 4-heptanone, SVM, ivermectin (Ivr) and aldicarb (Ald) on WT and mutant strains of C. elegans.
(A) WT, (B) ivermectin-resistant mutant DA1316, (C) aldicarb-resistant mutant CB113. L4 nematodes were suspended in 0.5 mL 0.01 M MES in vials lined up in a sealed 1-L box. The indicated concentration of the respective drug was added to the vials containing nematodes. The nematodes were harvested after 2 days, passed through a sieve and counted. Data on the y-axis are presented as percent (mean ± SE) mortality with respect to controls. There were 10 replicates for each treatment, with 300 nematodes per replicate. Graphs are representative of three independent experiments. Pairwise comparison of the means was performed by analysis of variance followed by post-hoc Tukey–Kramer multiple comparison test. Different letters indicate significantly different values (P ≤ 0.05).
Fig 5.
Effects of 4-heptanone and SVM on nuclear localization of DAF-16::GFP in C. elegans strain TJ356.
L4 larvae from strain TJ356 were treated with 4-heptanone or SVM for 24 h. Representative images of DAF-16::GFP expression in (A) control TJ356 worms and worms exposed to 4-heptanone, and SVM. Three independent experiments with at least 10 nematodes per group were performed. Scale bar = 100 μm. (B) Quantification of DAF- 16::GFP. The y-axis denotes the average number of pixels in TJ356 nematodes measured after the treatment. Mean expression ± SE from 10 nematodes is shown. Pairwise comparison of the means was performed by analysis of variance followed by post-hoc Tukey–Kramer multiple comparison test. Different letters indicate significantly different values (P ≤ 0.05). (C) Effect of 4-heptanone and SVM on sub cellular distribution of DAF16::GFP. Nuclear translocation of DAF16::GFP was examined in approximately 50 worms for each condition. The worms were scored as cytosolic, intermediate, and nuclear, on the basis of localization of the DAF-16::GFP. Data are presented as mean ±SEM of the percentages of each phenotype.
Fig 6.
Effects of 4-heptanone and SVM on the expression of SOD-3 and GST-4 in C. elegans strain CF1553 (sod-3::GFP) and CL2166 (gst-4::GFP).
L4 larvae was treated with 4-heptanone or SVM for 24 h. Representative images showing (A) sod-3::GFP and (B) gst-4::GFP expression in control or 4-heptanone and SVM-treated nematodes. Three independent experiments with at least 10 nematodes per group were performed. Scale bar = 100 μm. Quantification of (C) sod-3::GFP and (D) gst-4::GFP expression. The y-axis denotes the average number of pixels in L4 nematodes measured after the treatment. Mean expression ± SE from 10 nematodes is shown. Pairwise comparison of the means was performed by analysis of variance followed by the post-hoc Tukey–Kramer multiple comparison test. Different letters indicate significantly different values (P ≤ 0.05).
Fig 7.
Effects of 4-heptanone and SVM on the expression of SKN-1::GFP and gcs-1::GFP in C. elegans strain LD1 and LD1171.
L4 larvae was treated with 4-heptanone or SVM for 24 h. Representative images showing (A) SKN-1::GFP expression in control nematodes (B) Magnified view of the boxed region showing the ASI neurons (line arrow) in head region of control nematode (C) SKN-1::GFP localization in VOC treated nematodes (D) Magnified view of the boxed region in treated nematodes showing expression of SKN-1::GFP in intestinal nuclei (indicated by solid white triangles) (E) Effect of 4-heptanone and SVM on nuclear localization of SKN-1::GFP. Nuclear localization of SKN-1::GFP was scored as “Low", "Medium" or "High”. “High” refers to a strong GFP signal observed in more than 15 intestinal nuclei, "Medium" refers to nuclear GFP signals detected in 5–15 intestinal nuclei and "Low" indicates that SKN-1::GFP is hardly detectable in the intestinal nuclei. Data are presented as mean ±SEM (n = 50 worms per condition) of the percentages of each phenotype One way ANOVA and Tukey–Kramer multiple comparison test were performed independently for each localization phenotype. Different letters indicate significantly different values (P ≤ 0.05). (F) gcs-1::GFP expression in control or 4-heptanone and SVM-treated nematodes. Three independent experiments with at least 10 nematodes per group were performed. Scale bar = 100 μm. (G) Quantification of gcs-1::GFP expression. The y-axis denotes the average number of pixels in L4 nematodes measured after the treatment. Mean expression ± SE from 10 nematodes is shown. Mann-Whitney test indicated that fluorescence intensity in LD1171was significantly higher for 4-heptanone treatment when compared to control (p<0.001).