Lysine deficiency impaired growth performance and immune response and aggravated inflammatory response of the skin, spleen and head kidney in grown-up grass carp (Ctenopharyngodon idella)

Abstract

This dissertation was primarily focused on the immune response, inflammatory response and molecular mechanisms in the skin, head kidney and spleen of grown-up grass carp (Ctenopharyngodon idella). Six iso-nitrogen diets differing in lysine concentrations (5.6, 8.5, 11.6, 14.4, 17.5 and 20.7 g/kg) were fed to 540 grass carp (164.85 ± 0.79 g) for 60 d. After that, grass carp were challenged by Aeromonas hydrophila for 6 d. This study revealed that lysine deficiency (1) suppressed the growth performance of the fish and decreased their ability to resist skin lesion morbidity, (2) impaired the immune organ^'s immune response by decreasing the gene expressions of mucin, liver-expressed antimicrobial peptide (LEAP)-2B, β-defensin-1 and LEAP-2A and the production of antibacterial compounds of grown-up grass carp, and (3) aggravated the inflammatory response of immune organs in the fish by increasing the gene expressions of pro-inflammatory cytokines (interferon γ2 [IFN-γ2], tumor necrosis factor α [TNF-α], interleukin [IL]-15, IL-17D, IL-12p40, IL-6 and IL-8) and down-regulating anti-inflammatory cytokines (IL-11, transforming growth factor β1 [TGF-β1], IL-10 and IL-4/13A), which were tightly correlated with signal transducer and activator of transcription (STAT)1 and STAT3 signaling pathway, respectively. The different phenomenon in the skin, spleen and head kidney of fish may be correlated with the difference in gene subtype. In addition, using quadratic regression analysis of percent weight gain (PWG), skin lesion morbidity, and the lysozyme activities in the spleen and head kidney, the dietary lysine requirements for grown-up grass carp were estimated to be 13.58, 13.51, 14.56 and 14.18 g/kg, respectively.

Keywords: Grass carp (Ctenopharyngodon idella); Head kidney; Immune function; Lysine deficiency; Skin; Spleen.

Fig. 1

Quadratic regression analysis of percent weight gain (PWG) (A), skin lesion morbidity (B) and lysozyme activity in head kidney (C) and spleen (D) for grown-up grass carp fed diets containing graded levels of lysine (g/kg) for 60 d.

Fig. 2

Effects of dietary lysine (g/kg) on the skin hemorrhage and lesion morbidity of grown-up grass carp (Ctenopharyngodon idella) after infection of Aeromonas hydrophila. (A) Low or excess levels of dietary lysine led to obvious skin haemorrhage and lesions, compared to optimal dietary lysine level in on-growing grass carp (Ctenopharyngodon idella). (B) The skin haemorrhage and lesion morbidity in grown-up grass carp (Ctenopharyngodon idella). Data represent means of 24 fish in each group, error bars indicate S.D. All values are presented as the means ± SD, (n = 3 replicates, and each replicate with 8 fish). Values marked with different letters (a to e) are significantly different (P < 0.05).

Fig. 3

The histological observation in the head kidney and spleen of grown-up grass carp (Ctenopharyngodon idella) fed diets containing graded levels of lysine for 60 d, and then infected with Aeromonas hydrophila for 6 d. (A) The head kidney of un-supplemented group. (B) The head kidney of 14.4 g/kg diet group. (C) The head kidney of 20.7 g/kg diet group. (D) The spleen of un-supplemented group. (E) The spleen of 14.4 g/kg diet group. (F) The spleen of 20.7 g/kg diet group. V: vacuolization; H: hemosiderin; BS: blood vessel wall incrassating and endothelial cell swelling and defluxion; M: macrophage; EB: extravasated blood. The sections were stained with haematoxylin and eosin (H & E) and observed at 400× magnification.

Fig. 4

Heat-map of the mRNA levels of antimicrobial peptides, cytokines, STAT signalling pathway-related molecules in the spleen, head kidney and skin of grown-up grass carp fed graded levels of lysine. LEAP-2 = liver expressed antimicrobial peptide 2; IL = interleukin; TNF-α = tumor necrosis factor α; IFN-γ2 = interferon γ2; TGF-β = transforming growth factor β; TOR = target of rapamycin; TYK2 = tyrosine kinase 2; STAT = signal transducers and activators of transcription; JAK2 = Janus kinase 2. The values of upregulation (red) and downregulation (green) (i.e. from 1.1 to −1.0) represent log2 fold changes, as compared to group 1. n = 6 for each lysine level. The fold changes is based on the mRNA expression levels of different genes.

Fig. 5

Western blot analysis of signal transducers and activators of transcription 3 (STAT3) phosphorylation at Tyr705 proteins in the spleen (A), head kidney (B) and skin (C) of grown-up grass carp fed diets containing graded levels of lysine for 60 d, and then infected with Aeromonas hydrophila for 6 d. Data represent means (n = 3 replicates in each group), error bars indicate SD. All values are presented as the means ± SD. Values marked with different letters (a to d) are significantly different (P < 0.05).

Fig. 6

The potential pathways about the effects of lysine on immune function in the head kidney, spleen and skin of fish. TYK2 = tyrosine kinase 2; JAK2 = Janus kinase 2; STAT = signal transducers and activators of transcription; LZ = lysozyme; C3 = complement 3; C4 = complement 4; IgM= immunoglobulin M; TNF-α = tumor necrosis factor α; IFN-γ2 = interferon γ2; IL = interleukin; TGF-β = transforming growth factor β.