Mori T et al. (2023), Novel predator-induced phenotypic plasticity by...

XB-ART-59823

Front Physiol 2023 Jan 01;14:1178869. doi: 10.3389/fphys.2023.1178869.

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Novel predator-induced phenotypic plasticity by hemoglobin and physiological changes in the brain of Xenopus tropicalis.

Mori T, Machida K, Kudou Y, Kimishima M, Sassa K, Goto-Inoue N, Minei R, Ogura A, Kobayashi Y, Kamiya K, Nakaya D, Yamamoto N, Kashiwagi A, Kashiwagi K.

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Genes referenced: pigy

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	FIGURE 1. Experimental design and multiscale analysis. (A) Experimental design. Control represents no predator exposure. Ex 10 days, Ex 6, Ex 24, and Ex 48 h represent that one salamander was reared with 20 Xenopus tropicalis tadpoles in an aquarium for 10 days, 6, 24, and 48 h before sampling, respectively. Ex 5 days-Out represents that the salamander was removed after 5 days, and that the tadpoles were reared for the next 5 days without predation threat. Red arrows represent the period that the salamander was present. Each treatment group had three replicate aquaria. (B) Body parameters of the tadpoles measured for principal component analysis. Body weight was measured as parameter 1. Data were analyzed using ImageJ (ver1.53e: https://imagej.nih.gov/ij/index.html). (C) Results of the averaged PCA. Ex 10 days, Ex 48, Ex 24, Ex 5 days-Out, and control are shown as 10 days, 48, 24 h, 5 days out, and C, respectively. X-axis shows the main measurement factors chosen for the first principal component (numbers 4, 9, 10, 12, 13, 14, and 15; see Figure 2B). y-axis shows the main measurement sites selected for the second component (2, 3, 6, 7, and 8). The bars on the x-axis and y-axis for each sample indicate the standard error. multivariate analysis of variance (MANOVA) was performed on the principal component analysis (PCA) results. * denotes statistically significant (p < 0.05) differences for both the first and second components. Error bars indicate standard errors. See Supplementary Methods and Supplementary Table S4 for detailed information.
	FIGURE 2. (Continued).
	FIGURE 3. Effects of predation stress on energy production systems. (A) Metabolites and enzymes related to glycolysis, the tricarboxylic acid cycle (TCA), and ß-oxidation in the brain of Xenopus tropicalis tadpoles subjected to predator stress. Ingenuity pathway analysis (IPA) (Content v.48207413) was used to create Figure 4A. Concentration of metabolites and gene expression levels are examined in (B). Numbers ①–⑦ in (A) correspond to those shown in (B). Ex 10 days, Ex 24, Ex 6 h, and Control are shown as 10 D, 24, 6 h, and C, respectively. CE–MS and Dri-Chem systems were used to measure glucose content in brain tissue and body fluids, respectively. (C) Predictions of changes in the expression of fatty acid-binding proteins (fabp1–9), long-chain fatty-acid-CoA synthase (acsl), and carnitine O-palmitoyltransferase (cpt) type I–II using IPA. Blue and red represent down- and upregulation, respectively; green and pink represent down- and upregulation in RNA-seq data, respectively; blue and orange arrows represent down- and upregulation, respectively. * p < 0.05 represented statistical significance based on one-way ANOVA, followed by a Bonferroni post hoc test or Dunnett’s T3 test.
	FIGURE 4. Capillary electrophoresis–mass spectrometry (CE–MS) analysis. (A) GSH and GSSG in Xenopus tropicalis brains were measured using CE–MS analysis, and the GSH/GSSG ratio was determined. SOD3 expression was determined using real-time PCR, whereas reactive oxygen species (ROS) were measured using the d-ROMs test. (B) Illustration of the glutathione oxidation-reduction cycle. (C) Val, Leu, and Ile were measured using CE–MS analysis. Ex 10 days, Ex 48, Ex 24, Ex 6 h, and control are shown as 10 D, 48, 24, 6 h, and C, respectively. *p < 0.05 represented statistical significance based on one-way ANOVA, followed by a Bonferroni post hoc test or Dunnett’s T3 test.
	FIGURE 5. Analysis of hemoglobin using a hyperspectral camera (A) Randomly selected tadpoles were analyzed using a hyperspectral camera. The red signal indicates hemoglobin intensity. (B) The signal intensity of hemoglobin was determined by averaging the luminance of the area with high intensity (n = 2). Data were analyzed using ImageJ (ver1.53e: https://imagej.nih.gov/ij/index.html). (C) Oxygen–hemoglobin measurements and C–8D are enlargements of the area surrounded by red dots. (D) Oxygen–hemoglobin analysis in the brain was conducted using a similar method to that described in (B). The number of samples used for the control, 6, 24, 48 h, and 10 days groups were four, three, four, three, and two, respectively. Data were analyzed using ImageJ (ver1.53e). Ex 10 days, Ex 48, Ex 24, Ex 6 h, and control are shown as 10 D, 48, 24, 6 h, and C, respectively. *p < 0.05 represented statistical significance based on one-way ANOVA, followed by a Bonferroni post hoc test or Dunnett’s T3 test.
	FIGURE 6. Immunohistochemistry of brains from Xenopus tropicalis tadpoles exposed to predation stress. (A) Tadpole brains stained using anti-HBE1 antibody (red). Images were taken using an overlay of constant Z-stack height, under a confocal laser-scanning microscope, with a total of 35 photos in the stack. Negative control (Ng) represents the absence of a primary antibody and the presence of a secondary antibody: control (cont), 6 h (Ex 6 h), 24 h (Ex 24 h), 10 days (Ex 10 days). Parts of the brain shown: (I) around the telencephalon, (II) lateral regions of the diencephalon and mesencephalon; (III) medial zone of the mesencephalon; and (IV) medial zone of the medulla oblongata. (B) Measurement of HBE1 intensity in the metencephalon, shown by the dotted yellow line. TC, DC, MS, and MT represent the telencephalon, diencephalon, mesencephalon, and metencephalon, respectively. (C) The signal intensity of hemoglobin in MT was determined by averaging the luminance of the area with high intensity . Xenopus tropicalis tadpoles were used in this experiment (n = 6). Data were analyzed using ImageJ (ver1.53e: https://imagej.nih.gov/ij/index.html). Kolmogorov–Smirnov test showed that the data were normally distributed (p = 0.723), whereas Levene’s test showed that the assumed equality of error variance had a significance of 0.05 (p = 0.160). *p < 0.05 represented statistical significance based on one-way ANOVA, followed by a post hoc comparison using Dunnett’s T3.
	FIGURE 7. Ingenuity pathway analysis of genes expressed in the brain of Xenopus tropicalis. (A) Heat map of the signal transduction pathways in each treatment group, where 6 h/C, 24 h/C, 48 h/C, 10 days/C, and 5 days-O/C represent gene expression in the brains of Ex 6, Ex 24, Ex 48 h, Ex 10 days, and Ex 5 days-Out tadpoles divided by that in the control, respectively. Red, blue, and white represent upregulation, downregulation, and no change compared with the control, respectively. (B) Disease and function predicted by IPA based on gene expression compared with those in the control and Ex 10 days. Blue and red represent up- and downregulation, respectively. (C) Prediction of RhoA signaling based on IPA (Content v.48207413). Microtubule dynamics, branching neurons, and neuritogenesis were predicted according to the comparisons used in (D).

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