Season affects the estrogen system and the immune response of common carp

Abstract

The physiology of ectothermic animals, including fish, is strictly regulated by season-related external factors such as temperature or photoperiod. The immune response and the production of hormones, such as estrogens, are therefore also subject to seasonal changes. This study in common carp aimed to determine how the season affects the estrogen system and the immune response, including the antibacterial response during Aeromonas salmonicida infection. We compared the immune reaction in spring and autumn in the head kidney and liver and found that carp have higher levels of blood 17β-estradiol in autumn, while in the liver of these fish there is a higher constitutive expression of genes encoding vitellogenin, estrogen receptors and Cyp19 aromatase than in spring. Fish sampled in autumn also exhibited higher expression of immune-related genes in the liver. In contrast, in the head kidney from fish sampled in the autumn, the expression of genes encoding estrogen receptors and aromatase was lower than in spring, and a similar profile of expression was also measured in the head kidney for inos, arginases and il-10. In turn, during bacterial infection, we observed higher upregulation of the expression of inos, il-12p35, ifnγ-2, arginase 2 and il-10 in the liver of carp sampled in spring. In the liver of carp infected in spring a higher upregulation of the expression of the genes encoding CRPs was observed compared to fish infected during autumn. The opposite trend occurred in the head kidney, where the upregulation of the expression of the genes involved in the immune response was higher in fish infected in autumn than in those infected in spring. During the infection, also season-dependent changes occurred in the estrogen system. In conclusion, we demonstrated that season differentially affects the estrogenic and immune activity of the head kidney and liver. These results reinforce our previous findings that the endocrine and immune systems cooperate in maintaining homeostasis and fighting infection.

Keywords: Common carp; Estrogen system; Head kidney; Immune response; Liver; Seasonality.

Fig. 1

Season-dependent differences in the estrogen system. a—level of E2 in blood plasma, b—gene expression of vitellogenin in the liver, c, d—gene expression of estrogen receptors (erα, erβ and gper1) and enzymes involved in E2 conversion (cyp19a and b) in the head kidney (HK) and liver. Constitutive gene expression was determined by quantitative RT-qPCR and expressed relative to the expression of the 40S ribosomal protein s11 gene. Averages and SE (n=5–7). Stars indicates significant differences between autumn (orange bar) and spring (green bar) (* p ≤ 0.05, ** p ≤ 0.01)

Fig. 2

Season-dependent differences in the constitutive expression of inflammatory mediators in the head kidney (a, b—HK) and liver (c—e). Constitutive gene expression of proinflammatory (inos, il-1β, il-12p35, ifn-γ2), anti-inflammatory (arginase 1, arginase 2, il-10) mediators and acute phase proteins (c3, crp1, crp2) was determined by quantitative RT-qPCR and expressed relative to the expression of the 40S ribosomal protein s11 gene. Averages and SE (n=5–7). Stars indicates significant differences between autumn (orange bar) and spring (green bar) (* p ≤ 0.05, ** p ≤ 0.01)

Fig. 3

Season-dependent differences in the gene expression of inflammatory markers in the head kidney of Aeromonas salmonicida infected fish. Fish were i.p. injected with A. salmonicida (4 × 10⁸ bacteria in 250 μl PBS per fish). At 24 and 96 h post-infection (hpi) the head kidneys were collected, and gene expression of proinflammatory (a—inos, b—il-1β, c—il-12p35, d—ifn-γ2) and anti-inflammatory (e—arginase 1, f—arginase 2, g—il-10) mediators was measured by quantitative RT-qPCR. Averages and SE (n=5–7). Changes in gene expression are shown as x-fold increase compared to the control group without infection (CTR) and standardized for the housekeeping gene 40S ribosomal protein s11. Mean values not sharing letters are statistically different between autumn (orange bar) and spring (green bar)

Fig. 4

Season-dependent differences in the gene expression of inflammatory markers in the liver in Aeromonas salmonicida infected fish. Fish were i.p. injected with A. salmonicida (4 × 10⁸ bacteria in 250 μl PBS per fish). At 24 and 96 h post-infection (hpi) the livers were collected, and gene expression of proinflammatory (a—inos, b—il-1β, c—il-12p35, d—ifn-γ2), anti-inflammatory (e—arginase 1, f—arginase 2, g—il-10) mediators was measured by quantitative RT-qPCR. Averages and SE (n=5-7). Changes in gene expression are shown as x-fold increase compared to the control group without infection (CTR) and standardized for the housekeeping gene 40S ribosomal protein s11. Mean values not sharing letters are statistically different between autumn (orange bar) and spring (green bar)

Fig. 5

Season-dependent differences in the gene expression of acute phase proteins in the liver in Aeromonas salmonicida infected fish. Fish were i.p. injected with A. salmonicida (4 × 10⁸ bacteria in 250 μl PBS per fish). At 24 and 96 h post-infection (hpi) the livers were collected, and gene expression of acute phase proteins (a—c3, b—crp1, c—crp2) was measured by quantitative RT-qPCR. Averages and SE (n=5–7). Changes in gene expression are shown as x-fold increase compared to the control group without infection (CTR) and standardized for the housekeeping gene 40S ribosomal protein s11. Mean values not sharing letters are statistically different between autumn (orange bar) and spring (green bar)

Fig. 6

Season-dependent and infection-induced changes in estrogen system in the head kidney. Fish were i.p. injected with A. salmonicida (4 × 10⁸ bacteria in 250 μl PBS per fish). At 24 and 96 h post-infection (hpi) the head kidneys were collected, and gene expression of estrogen receptors (a—erα, b—erβ and c—gper1) and enzymes involved in E2 conversion (d—e cyp19a and b) was measured by quantitative RT-qPCR. Averages and SE (n=5–7). Changes in gene expression are shown as x-fold increase compared to the control group without infection (CTR) and standardized for the housekeeping gene 40S ribosomal protein s11. Mean values not sharing letters are statistically different between autumn (orange bar) and spring (green bar)

Fig. 7

Season-dependent and infection-induced changes in the expression of estrogen system genes in the liver. Fish were i.p. injected with A. salmonicida (4 × 10⁸ bacteria in 250 μl PBS per fish). At 24 and 96 h post-infection (hpi) the livers were collected, and gene expression of estrogen receptors (a—erα, b—erβ and c—gper1) and enzymes involved in E2 conversion (d—e, cyp19a and b) was measured by quantitative RT-qPCR. Averages and SE (n=5–7). Changes in gene expression are shown as x-fold increase compared to the control group without infection (CTR) and standardized for the housekeeping gene 40S ribosomal protein s11. Mean values not sharing letters are statistically different between autumn (orange bar) and spring (green bar)