Fig. 3: ROS levels are induced by parasitisation and important for escape behaviour. | Nature Communications

Fig. 3: ROS levels are induced by parasitisation and important for escape behaviour.

From: Neofunctionalization of an ancient domain allows parasites to avoid intraspecific competition by manipulating host behaviour

Fig. 3

a Representative images of the CNSs of nonparasitized control (CK) and Lb-parasitized larvae (Parasitized). The CNS was stained with DCFH-DA. Scale bars: 50 μm. b ROS levels as a function of time in larval CNS after exposure to female Lb wasps (n = 3 replicates, at least seven Drosophila CNSs were examined for each individual). The dashed line indicates the ROS levels of the nonparasitized larvae, which was used as a control. Data are presented as mean values ± SD, and significance was analysed using one-way ANOVA followed by Šidák’s multiple-comparison test (1 h: P = 6.2 × 10−8; 2 h: P = 6.5 × 10−6; 3 h: P = 0.001; 4 h: P = 0.7629; 5 h: P = 0.9999; 6 h: P = 0.9999; 12 h: P = 0.9999; 24 h: P = 0.9999; **P < 0.01; ***P < 0.001; ns: not significant). c Representative images of CNSs from 1× PBS-injected control (PBS) and Lb venom (1:50 dilution)-injected larvae (Venom). The CNS was stained with DCFH-DA. Scale bars: 50 μm. d ROS levels as a function of time in larval CNS after injection with female Lb venom at a 1:50 dilution. The dashed line indicates the ROS levels of the 1×PBS-injected larvae, which was used as a control (n = 3 replicates, at least seven Drosophila CNSs were examined for each individual). Data are presented as mean values ± SD, and significance was analysed by one-way ANOVA followed by Šidák’s multiple-comparison test (1 h: P = 0.0002; 2 h: P = 4.8 × 10−5; 3 h: P = 0.0307; 4 h: P = 0.9998; 5 h: P = 0.8802; 6 h: P = 0.9999; 12 h: P = 0.9999; 24 h: P = 0.9949; *P < 0.05; ***P < 0.001; ns: not significant). e Representative images of CNSs from Lb-parasitized larvae with or without expression of catalase from ubiquitously expressed Actin-GAL4, neuron-specific Elav-GAL4 or haemocyte-specific Hml-GAL4. The CNS was stained with DCFH-DA. Scale bars: 50 μm. f Quantification of ROS levels in the CNSs of larvae with or without expression of UAS-Catalase using different Actin-GAL4 for ubiquitous expression, Elav-GAL4 for expression in neurons and Hml-GAL4 for expression in haemocytes. Plotted is the mean intensity from fifty areas within each CNS (n = 12 Drosophila CNSs were examined for each individual). Data are presented as mean values ± SD. Significance was determined by two-sided unpaired Student’s t test (WT: P = 0.4986; Actin-GAL4: P = 2.0 × 10−13; Elav-GAL4: P = 1.0 × 10−15; Hml-GAL4: P = 0.8557; ***P < 0.001; ns: not significant). g Escape indices for Drosophila hosts of the indicated genotypes. The number of larvae that exhibited escape behaviour was recorded from 30 min to 75 min in the presence of parasitoids. Left to right: n = 288, 250, 368, 238, 235, 403, 448 and 238. At least three biological replicates were performed. Data are presented as mean values ± SD. Significance was determined by two-sided unpaired Student’s t test (WT: P = 0.7217; Actin-GAL4: P = 0.0004; Elav-GAL4: P = 3.7 × 10−5; Hml-GAL4: P = 0.5762; ***P < 0.001; ns: not significant). h Western blot analysis of EsGAP1 in the parasitoid venom apparatus and carcass. The representative images out of three independent replicates are displayed. CBB: Coomassie Brilliant Blue. i Representative images of EsGAP1 (red) immunolocalization in the nonparasitized larval brain (CK) and parasitized host brain (parasitized) 1 h after exposure to female Lb. Nuclei stained with DAPI (blue). Scale bars: 20 μm. j Characterised EsGAP1-staining spots in the CNS of parasitized and nonparasitized hosts (n = 20 biologically independent samples were examined for each group). Data are presented as mean values ± SD. Significance was determined by two-sided unpaired Student’s t test (P = 1.0 × 10−15; ***P < 0.001). k Representative CNS image of UAS-EsGAP1 or Elav-GAL4 > UAS-EsGAP1 larva stained with DCFH-DA. Scale bar: 50 μm. l Quantification of ROS levels in the CNSs of UAS-EsGAP1 or Elav-GAL4 > UAS-EsGAP1 larvae. Each plot indicates the mean intensity from fifty areas within each CNS (n = 10 per phenotype). Data are presented as mean values ± SD. Significance was determined by two-sided unpaired Student’s t test (P = 1.0 × 10−15; ***P < 0.001). m Representative images of CNS from larvae parasitized by dsGFP-treated or dsEsGAP1-treated female Lb. The CNS was stained with DCFH-DA. Scale bars: 50 μm. n Quantification of ROS levels in the CNSs of larvae that parasitized by dsGFP-treated or dsEsGAP1-treated female Lb. Each plot indicates the mean intensity from fifty areas within each CNS (n = 10 per group). Data are presented as mean values ± SD. Significance was determined by two-sided unpaired Student’s t test (P = 3.6 × 10−13; ***P < 0.001). Source data are provided as a Source data file.

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