Fig 1.
Atlantic croaker sampling sites and brain hif-α mRNA levels in croaker collected from hypoxic and normoxic sites in the nGoM.
(A) Location of four hypoxic sites (C8, C9, F3 and F4) in the coastal region in the nGoM and two normoxic reference sites (N1 and N2) to the east of the Mississippi Delta where croaker were collected in August, 2007; July, 2008; and August, 2012. The map including sampling sites was generated using Ocean Data View software. Black circles indicate sampling sites. Insert bar graphs indicate dissolve oxygen (DO: mg l-1) levels during sampling period. (B, C) Relative hif-1α (B) and hif-2α (C) mRNA levels in brains of croaker collected from normoxic (N1, N2) and hypoxic (F3, F4, C8, C9) sites in August 2007 (i), July 2008 (ii), and August 2012 (iii) in the nGoM. Asterisks denote significant differences between normoxic (reference) and hypoxic sites (nested ANOVA, **p<0.01). The horizontal lines represent mean values, N = 8. Individual site differences are indicated with different letters (Fisher’s PLSD, p<0.05). DO, dissolved oxygen (mg l-1).
Fig 2.
Expression of hif-1α and hif-2α mRNA levels in brains of Atlantic croaker exposed to laboratory hypoxia.
Effects of 7-day laboratory exposure to normoxia control (CTL, dissolved oxygen, DO: ~6.5 mg l-1, white bars), hypoxia (HYP, DO: 1.7 mg l-1, black bars) and a recovery period on relative hif-1α (A-ii) and hif-2α (B-ii) mRNA levels, and (C) Ct values of 18S rRNA in croaker brains. Note: here and in subsequent figures laboratory exposure duration only refers to period fish were exposed to target DO; fish were previously exposed to declining DO for additional 2-day adjustment period. After 7-day hypoxia exposure, DO of hypoxic treatment was restored to normoxic level (24-h recovery period). Each bar represents the mean±SEM (N = 7–8). Differences in the relative mRNA levels between the start of the experiment (CTL, control) and each treatment were tested by Dunnett’s test, *p<0.05. ‘†’ indicates significant difference from normoxic controls (Student’s t-test, p<0.05). Ct, threshold cycle.
Fig 3.
Brain nNOS mRNA and protein levels in Atlantic croaker collected from hypoxic and normoxic sites in the nGoM.
Relative nNOS mRNA levels (A), (B) Ct values of 18S rRNA, and nNOS protein expression and levels (C,D) in brains of croaker collected from normoxic (N1, N2) and hypoxic (F3, F4, C8, C9) sites in August 2007 (i), July 2008 (ii), and August 2012 (iii). Representative Western blot of nNOS protein expression in croaker brain samples (B). The horizontal lines represent mean values, N = 8. A nested ANOVA indicates nNOS mRNA and protein levels in croaker from the normoxic sites were significantly different from those in fish from the hypoxic sites (**p<0.01). Individual site differences identified with a multiple range test, Fisher's PLSD, are indicated with different letters. M, marker; kDa, kilodalton.
Fig 4.
Expression of nNOS mRNA in the brains and plasma NOx levels in Atlantic croaker exposed to laboratory hypoxia.
Effects of 7-day laboratory exposure to normoxia (dissolved oxygen, DO: ~6.5 mg/L, white bars), hypoxia (HYP, DO: 1.7 mg l-1, black bars) and a recovery period on hypothalamic nNOS mRNA levels (A) and plasma NO metabolites, nitrite plus nitrate (NOx) levels (B). Each value represents the mean±SEM (N = 7–11). Differences in the relative mRNA levels between the start of the experiment (CTL, control) and each treatment were tested by Dunnett’s test, *p<0.05.
Fig 5.
igfbp-1 mRNA levels in livers of Atlantic croaker collected from normoxic and hypoxic sites in the nGOM and after exposure to hypoxia in the laboratory.
Croaker collected from normoxic (N1, N2) and hypoxic (F3, F4, C8, C9) sites in August 2007 (i), July 2008 (ii), and August 2012 (iii) in the nGoM (A). Asterisks denote significant differences between normoxic (reference) and hypoxic sites (nested ANOVA, **p<0.01). The horizontal lines represent mean values, N = 7–8. Individual site differences are indicated with different letters (Fisher’s PLSD, p<0.05). (B, C) Expression of igfbp-1 mRNA levels and Ct values of 18S rRNA in livers of croaker exposed to laboratory hypoxia. Effects of 7-day laboratory exposure to normoxia (DO: ~6.5 mg l-1, white bars), hypoxia (HYP, DO: 1.7 mg l-1, black bars) and recovery period on igfbp-1 mRNA levels in croaker liver. Each value represents the mean±SE (N = 7–8). Differences in the relative mRNA levels between the start of the experiment (CTL, control) and each treatment were tested by Dunnett’s test, *p<0.05. ‘†’ indicates significant difference from normoxic controls (Student’s t-test, p<0.05).
Fig 6.
Hierarchical clustering of gene expression in the brain and liver tissues of Atlantic croaker exposed to environmental and laboratory hypoxia.
Croaker collected from normoxic (N1, N2) and hypoxic (C8, C9) sites in August 2007 and July 2008 in the nGoM (A) and laboratory hypoxia experiment (B). A: August; J: July.
Fig 7.
Protein carbonyl (PC) contents in Atlantic croaker exposed to environmental and laboratory hypoxia.
(A) PC contents in croaker liver collected from normoxic (N1, N2) and hypoxic (F3, F4, C8, C9) sites in August 2007 (i), July 2008 (ii) and August 2012 (iii) in the nGoM. The horizontal lines represent mean values, N = 8. A nested ANOVA indicates PC contents in croaker liver from the normoxic sites were significantly different from those in fish from the hypoxic sites (**p<0.01). Individual site differences identified with a multiple range test, Fisher's PLSD, are indicated with different letters. DO, dissolved oxygen (mg l-1). (B) Effects of 7-day laboratory exposure to normoxia (DO: ~6.5 mg l-1, white bars), hypoxia (HYP, DO: 1.7 mg l-1, black bars) and recovery period on PC contents in croaker liver. Each value represents the mean±SE (N = 7–8). Differences in the PC contents between the start of the experiment (CTL, control) and each treatment were tested by Dunnett’s test, *p<0.05. prot., protein. (C) Proposed model of hypoxia-induced upregulation of croaker hif-α, nNOS, igfbp transcripts, NOx, and ROS and RNS (solid arrows pointing up), based on our present and previous studies (1,2,3Rahman and Thomas, 2007, 2011, 2013). The model shows several pathways (dotted lines) through which hypoxia could potentially upregulate gene and biochemical biomarkers in croaker. ‘?’: evidence has only been obtained in mammalian in vitro studies (Li and Jackson, 2002).