A 5' Promoter Region SNP in CTSC Leads to Increased Hypoxia Tolerance in Changfeng Silver Carp (Hypophthalmichthys molitrix)

Abstract

Silver carp is a critically significant species in freshwater aquaculture in China, characterized by its limited tolerance to hypoxia. In this study, a significant SNP locus at Chr8: 29647765 (T/C) associated with hypoxia tolerance traits was identified in Changfeng silver carp, and the homozygotic CC genotype exhibited higher hypoxic tolerance than the homozygotic TT and heterozygotic TC genotypes. Under hypoxic conditions, the hemoglobin concentration increased, with the CC genotype demonstrating a significantly higher level compared with the TT genotype; the activities of antioxidant enzymes including catalase and superoxide dismutase were significantly higher in the CC genotype than in the other genotypes; the area of the gill lamellae was significantly smaller in the CC genotype than in the TT and TC genotypes; and the number of apoptotic cells in the brain was significantly lower in the CC genotype than in the TT and TC genotypes. Sequence analysis showed that this SNP was located in the promoter region of the cathepsin C (CTSC) gene. The expression levels of the CTSC gene were analyzed across the three genotypes, revealing that the CC genotype exhibited significantly lower expression compared with the TT and TC genotypes under hypoxia. This finding suggests that the SNP associated with the CC genotype leads to reduced CTSC expression, which may facilitate better physiological adaptation to hypoxia. Analysis of the promoter region of CTSC found a unique predicted hypoxia-inducible factor 1-alpha (HIF-1α) binding site (CGTG) in the T genotype, implying that the differential expression of CTSC among the three genotypes under hypoxic stress may be regulated by HIF-1α, a transcription factor integral to hypoxia adaptation, thereby affecting hypoxia tolerance, which further affects the immune response of the Changfeng silver carp in response to the hypoxic environment. Although SNPs represent significant genetic determinants, their phenotypic effects are predominantly mediated through complex interactions within gene regulatory networks and environmental influences. This study identified an effective SNP site in Changfeng silver carp, providing valuable guidance for future selective breeding and the development of new hypoxia-tolerant varieties.

Keywords: CTSC; HIF-1α; Hypophthalmichthys molitrix; SNP; hypoxia.

Figure 1

Survival curves of individuals with the TT (blue), TC (orange), and CC (red) genotypes (n = 20) under hypoxic stress. There was a statistically significant difference between the TT genotype and TC genotype. * p < 0.05.

Figure 2

Alterations in red blood cell number (A), hemoglobin concentration (B), SOD activity (C), and CAT activity (D) induced by hypoxic conditions across individuals of three different genotypes. Data are shown as the mean ± SD (n = 3). Hy_0h refers to normoxia, indicating no exposure to hypoxia. Hy_24h denotes exposure to hypoxia (2 mg/L) for a duration of 24 h. Blue represents the TT genotype, orange represents the TC genotype, and red represents the CC genotype. Distinct letters denote statistically significant differences (p < 0.05) among the three genotypes at a single time point. Asterisks indicate statistically significant differences within the same genotype across two time points (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

Structural changes in gills induced by hypoxic conditions across the three distinct genotypes. The microstructural analysis of the gills from individuals of various genotypes was conducted using scanning electron microscopy (A) and HE staining (B), with scale bars located in the lower right-hand corners. (C) The surface area measurements of the gill lamellae in individuals of different genotypes following normoxic and hypoxic treatments. (D) The variations in interlamellar cell mass (ILCM) thickness among individuals of different genotypes under normoxic and hypoxic conditions. Green arrows denote the gill lamellae, while red arrows indicate the ILCM between two adjacent gill lamellae. Data are shown as the mean ± SD (n = 3). Hy_0h refers to normoxia, indicating no exposure to hypoxia. Hy_24h denotes exposure to hypoxia (2 mg/L) for a duration of 24 h. Blue represents the TT genotype, orange represents the TC genotype, and red represents the CC genotype. Distinct letters denote statistically significant differences (p < 0.05) among the three genotypes under hypoxic stress. Asterisks indicate statistically significant differences within the same genotype across two time points (** p < 0.01, *** p < 0.001).

Figure 4

TUNEL staining of the brains in the three genotypes after exposure to hypoxia. (A) Light microscope micrographs depict brain cell apoptosis after 24 h of hypoxia. Apoptotic cells are indicated by a brown coloration, whereas normal cells are represented by a light blue hue. The right panel presents a magnified view of the area enclosed within the red box on the left. (B) Fluorescence microscopy detection of brain cell apoptosis after 24 h under the hypoxic condition. Apoptotic cells are shown in green fluorescence, DAPI staining (blue) represents the nuclei. The white box located in the upper right corner represents an enlarged view of the area delineated by the dotted line. (C) The statistics of the apoptosis rate in the brain under a light microscope after 24 h of hypoxia. (D) The statistics of the apoptosis rate in the brain under fluorescence microscopy after 24 h of hypoxia. Data are shown as the mean ± SD (n = 3). Blue represents the TT genotype, orange represents the TC genotype, and red represents the CC genotype. Different letters indicate significant differences (p < 0.05).

Figure 5

Structural and expression characteristics of the CTSC gene. (A) Schematic representation of the CTSC gene architecture. Orange ovals represent core promoter regions, and green boxes denote exons. (B) Expression profiles of the CTSC gene across different tissues in silver carp. Data are shown as the mean ± SD (n = 3).

Figure 6

The expression analysis of the CTSC gene among the three genotypes under hypoxia stress. Changes in CTSC gene expression in the heart (A), brain (B), liver (C), and gill (D) in individuals exhibiting three distinct genotypes under both normoxic and hypoxic conditions. (E) The prediction of HIF-1α transcription factor binding sites in the upstream region of the core promoter of the CTSC gene. Hy_0h refers to normoxia, indicating no exposure to hypoxia. Data are shown as the mean ± SD (n = 3). Hy_24h denotes exposure to hypoxia (2 mg/L) for a duration of 24 h. Blue represents the TT genotype, orange represents the TC genotype, and red represents the CC genotype. Cyan boxes represent ACGTG sequences, red boxes represent CGTG sequences, and orange ovals represent core promoter regions. Distinct letters denote statistically significant differences (p < 0.05) among the three genotypes under hypoxic stress. Asterisks indicate statistically significant differences within the same genotype across two time points (** p < 0.01, *** p < 0.001).

Figure 7

Analysis of immune-related gene expression in the liver after hypoxia. Changes in GOT1 (A), LYZ (B), angpt2 (C), and IL-6R (D) gene expression under hypoxia stress. Data are shown as the mean ± SD (n = 3). Hy_0h refers to normoxia, indicating no exposure to hypoxia. Hy_24h denotes exposure to hypoxia (2 mg/L) for a duration of 24 h. Blue represents the TT genotype, orange represents the TC genotype, and red represents the CC genotype. Distinct letters denote statistically significant differences (p < 0.05) among the three genotypes under hypoxic stress. Asterisks indicate statistically significant differences within the same genotype across two time points (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 8

Change in MDA (A), Na⁺/K⁺-ATP (B), glycogen (C), and lactate (D) concentration under hypoxia stress. Data are the mean ± SD (n = 3). Hy_0h refers to normoxia, indicating no exposure to hypoxia. Hy_24h denotes exposure to hypoxia (2 mg/L) for a duration of 24 h. Blue represents the TT genotype, orange represents the TC genotype, and red represents the CC genotype. Distinct letters denote statistically significant differences (p < 0.05) among the three genotypes under hypoxic stress. Asterisks indicate statistically significant differences within the same genotype across two time points (* p < 0.05, ** p < 0.01, *** p < 0.001).