Epidermal oxysterols function as alarm substances in zebrafish

Abstract

Alarm substances signal imminent predation thread and enable anti-predation strategies. In shoaling fish, alarm cues diffuse from injured skins that induce intense fear and anti-predation behaviors in other members. While these "fear substances" are shown to be present in numerous fishes and thought to exist in roughly 8,000 Ostariophysan species, their chemical nature remains largely unknown. We posited that fish alarm cues comprise small compounds and induce specific behaviors characteristic of fish exposed to skin extracts. Using the behaviors as bioassays, we tracked the alarm function of zebrafish skin extract to two compounds, 24-methyl-5α-cholestane-3α,7α,12α,24,28-pentahydroxy 28-sulfate, an oxysterol sulfate, and 5α-cyprinol sulfate. At concentrations of less than one nanomolar, each compound induced anti-predator behaviors and increased cortisol levels in zebrafish. Their mixture, at the natural ratio, replicated the skin extract in eliciting the full suite of anti-predator behavior patterns. Our findings reveal a molecular mechanism whereby fish escape predation danger.

Keywords: Behavioral neuroscience; Biological sciences; Biomolecules; Neuroscience.

Graphical abstract

Figure 1

Bioassay-guided fractionation of alarm substance (A) Bioassay-guided fractionation to identify alarm substance components. Boxed fractions replicated the activity of skin extract (SKE) in inducing changes in four behavioral phenotypes, including (B) erratic movements duration (%), (C) time in bottom half (%), (D) freezing duration (%), and (E) latency to upper half (s). Fractions of successive separation by petroleum (PET), ethyl acetate (EtOAc), n-butanol (nBuOH), and fraction 7 of nBuOH, were reconstituted in water to equivalents of a 1000-fold dilution of SKE. Representative motion traces of females exposed to (F) vehicle, (G) SKE, (H) nBuOH, and (I) fraction 7, respectively. ∗p < 0.05; ∗∗p < 0.01. See Figure S2 for detailed fractionation procedures. In B-E, the exact sizes of biologically independent samples and the results of independent sample t-tests or Mann-Whitney U tests are summarised in Table S1. Data are represented as mean ± SEM.

Figure 2

Identification of alarm substance in the n-butanol extract (A) Chromatography of the n-butanol extract, which shows a predominant peak at retention time 7.05 min that includes 5α-cyprinol (5CS) and 5α-daniol sulfate (5DS) (insets, elucidated chemical structure, ion chromatogram, and mass spectrum, respectively). BPI, base peak ion; m/z, mass-to-charge ratio. (B and C) Key HMBC correlations (arrows) of putative analogs of 5DS. The dashed arrows represent unreasonable correlations.

Figure 3

Anti-predator behaviors induced by 5CS and 5DS with representative trajectories 5α-cyprinol (5CS) and 5α-daniol sulfate (5DS) each induced certain anti-predator behaviors: (A) erratic movement duration (%), (B) time in the bottom half (%), (C) freezing duration (%), and (D) latency to the upper half (s). The mixture of 5CS and 5DS at a ratio of 100:1 induced all four behaviors (E‒H; 5DS concentrations labeled on the X axis; 5CS concentrations are 100 times those of 5DS). Skin extract (SKE) was tested at a 1000-fold dilution as a positive control. Representative female motion traces triggered by (I) 10⁻¹⁰ M of 5CS, (J) 10⁻¹⁰ M of 5DS, and (K) a mixture of 5CS (10⁻⁸ M) and 5DS (10⁻¹⁰ M). ∗p < 0.05, ∗∗p < 0.01. Letters above the line indicate significant differences between groups and ∗ or ∗∗ indicate a difference in behaviors induced by different compounds at the same concentration. The exact sizes of biologically independent samples and results of ANOVA followed by Bonferroni multiple comparisons ((A) two-sided Kruskal–Wallis test (B and F) and Tamhane test (C, D, E, G, and H)) are summarised in Table S9. In addition, statistical results of behavior comparison caused by different compounds at a specific concentration are summarised in Table S10 with independent samples t-test (A, C, and D) and Mann-Whitney U test (B). Data are represented as mean ± SEM.

Figure 4

Whole-body cortisol level (ng g⁻¹ body mass) in adult zebrafish exposed to 5CS and 5DS at concentrations from 10⁻¹² M to 10⁻⁶ M Vertical bars, one SEM. For each compound, responses that do not share a letter are different. Asterisk indicates different cortisol levels induced by different compounds at the same concentration (∗p < 0.05; ∗∗p < 0.01). The exact sizes of biologically independent samples and the results of the two-sided Kruskal–Wallis test followed by a multiple comparison of mean ranks are summarised in Table S11. Independent samples t-test (concentration (lg[M]) = −12, −10 and −6) and Mann-Whitney U test (concentration (lg[M]) = −8) of the comparison at the same concentration are summarised in Table S12. Data are represented as mean ± SEM.