Thermal imprinting modifies adult stress and innate immune responsiveness in the teleost sea bream

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   Figure 1

   Schematic representation of the experimental setup. (A) Temperature program imposed to gilthead sea bream from egg fertilization to larvae-juvenile transition. Four temperature treatments (thermal groups) were considered: 2 constant temperatures (18–18°C (LT) and 22–22°C (HT)) and 2 variable temperatures between incubation phase and larval rearing (22–18°C (HLT) and 18–22°C (LHT)). When the fishes’ bodies were covered with scales (larvae-juvenile transition), fish from all thermal groups were maintained at 22°C until the onset of the acute stress challenge by confinement (n = 20/tank; 2 replicate tanks per thermal history group). Details about differences in the development of fish from different thermal regimes and the time of temperature change/group can be accessed in Supplementary Tables 1 and 2 and also in Supplementary Fig. 1B. Acute stress challenge by confinement and the sampling regime. Adult sea bream (n = 5/replicate tank) stocked at 20 kg/m³ were sampled prior to the application of an acute stress challenge (0 h). The remaining fish were exposed to an acute stress by reducing the level of water of each tank, which increased the density to 70 kg/m³. Fish were exposed to confinement stress for 30 min before returning them to the initial stocking density (20 kg/m³) and then sampled (n = 5/replicate tank/sampling time) at 1, 4 and 24 h after a stress challenge.
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   Figure 2

   Cortisol levels and expression of molecular indicators (pomc α1, pomc α2, gr and star) of the overall stress response of the four experimental thermal groups after acute stress confinement. Cortisol levels were Log10 transformed and measured before (0 h) and after acute stress at three time points (1, 4 and 24 h). Gene expression was assessed by qPCR at 0, 4 and 24 h, and in the pituitary gland, expression of pomcα1, pomcα2 and gr was normalized by the mean of rps18 expression, and in the head kidney, the relative gene expression of star and gr by the mean of β-actin expression. Normalized data were expressed as Log2 Fold Change relative to LT 18–18°C at 0 h. The results are plotted in Tukey box and whiskers graphs: LT (18–18°C); LHT (18–22°C); HT (22–22°C); HLT (22–18°C). ‘+’ represents the mean and dots the outliers (n = 10 individuals per group per sampling time). Different letters indicate statistical significant differences between the groups of the same sampling time (intergroup analysis). Capital letters under the graph of plasma cortisol indicate if significant differences exist between sampling times for each thermal group (intra-group analysis, ns = not significantly different). Groups with the same letter are not significantly different. Significant upregulation or downregulation relative to LT (18–18°C) at 0 h was denoted by: *P < 0.05, **P < 0.01, ***P < 0.001 using two-way ANOVA.
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   Figure 3

   Histological (A) and histomorphometric (B) analysis of gilthead sea bream MMCs in the head kidney before and 1, 4 and 24 h after the acute stress challenge. (A) MMCs in head kidney were stained with Alcian blue–Periodic Acid Schiff (PAS)–Orange G. Different thermal groups (LT (18–18°C); HLT (22–18°C); LHT (18–22°C); HT (22–22°C)) are represented. Erythrocytes enriched area is stained in orange, and lymphoid tissue is stained in purple. MMCs may consist in larger clusters of cells (area >0.001 mm²; arrowhead) or as free cells (area <0.001 mm²; arrow; e). MMCs may present a round (a), oval (c) or irregular shape (g) and is frequently observed near blood vessels (bv, e). Both free and larger MMCs contain dark brown pigmented material. ad, adipocytes. Scale bar = 100 µm. (B) Histomorphometric analysis of MMCs in a randomly selected area of 0.3 mm² in head kidney: number of MMCs with an area equal or inferior to 0.001 mm² (smaller MMCs), total number of MMCs found within the analyzed area and the total area (mm²) occupied by MMCs in the selected area. Analysis of MMCs was performed in unstressed fish (0 h) and during stress response (1, 4 and 24 h). Different thermal regimes (LT (18–18°C); LHT (18–22°C); HT (22–22°C); HLT (22–18°C)) are represented. Different letters indicate statistical difference between thermal groups of the same sampling time. Data shown as mean ± s.e.m.; two-way ANOVA; P < 0.05; n = 10. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-16-0610
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   Figure 4

   Relative expression of dct and csf1r in the head kidney before the onset of confinement (0 h) and during response (4 and 24 h) to stress. Transcript expression levels were obtained by qPCR and were normalized in relation to the mean of β-actin expression and normalized results were expressed as Log2 Fold Change calculated relative to LT 18–18°C at 0 h. The results are plotted in Tukey box and whiskers graphs: LT (18–18°C); LHT (18–22°C); HT (22–22°C); HLT (22–18°C) are represented. ‘+’ represents the mean and dots the outliers (n = 10 individuals per group per sampling time). Different letters indicate statistical significant difference between thermal groups of the same sampling time (intergroup analysis). Data are shown as mean ± s.e.m. (n = 10 per group) and significant upregulation or downregulation relative to LT (18–18°C) at 0 h was denoted by: *P < 0.05, **P < 0.01, ***P < 0.001 using two-Way ANOVA.

• © 2017 Society for Endocrinology