Seeing in the swamp: hydrogen sulfide inhibits eye metabolism and visual acuity in a sulfide-tolerant fish

Abstract

In fish, vision may be impaired when eye tissue is in direct contact with environmental conditions that limit aerobic ATP production. We hypothesized that the visual acuity of fishes exposed to hydrogen sulfide (H₂S)-rich water would be altered owing to changes in cytochrome c oxidase (COX) activity. Using the H₂S-tolerant mangrove rivulus (Kryptolebias marmoratus), we showed that a 10 min exposure to greater than or equal to 200 µM of H₂S impaired visual acuity and COX activity in the eye. Visual acuity and COX activity were restored in fish allowed to recover in H₂S-free water for up to 1 h. Since K. marmoratus are found in mangrove pools with H₂S concentrations exceeding 1000 µM, visual impairment may impact predator avoidance, navigation and foraging behaviour in the wild.

1. Introduction

Vision in most animals is important for interpreting the surrounding environment. The response to visual stimuli directs many critical behaviours (e.g. navigation, foraging and predator avoidance) that ultimately affect survival. Eye tissue is energetically demanding because rod and cone photoreceptors in the retina need ATP to generate membrane potentials, as well as synthesize, and transport proteins [1–4]. In aquatic animals, environmental perturbations that impact aerobic metabolism (e.g. hypoxia) may harm vision [5].

One abiotic factor that severely limits aerobic metabolism in aquatic animals is hydrogen sulfide (H₂S). H₂S reversibly binds to cytochrome c oxidase (COX) within the mitochondrial electron transport chain, inhibiting oxidative phosphorylation and decreasing aerobic ATP production [6–8], among other toxic effects [9]. Not surprisingly, most fishes are intolerant of H₂S [6], but a few species have survived and even speciated in H₂S-rich water [10,11]. Is vision jeopardized in fishes exposed to H₂S?

We tested the hypothesis that the visual acuity of fishes exposed to H₂S-rich water would be impaired owing to reduced COX activity in the eye tissue. We studied the H₂S-tolerant mangrove rivulus (Kryptolebias marmoratus) that inhabits O₂-poor, H₂S-rich pools (up to 1166 µM of H₂S) in mangrove forests [12]. We measured both visual acuity and COX activity in the eye of fish at various H₂S concentrations (100–400 µM) and followed recovery after exposure.

2. Methods

(a) . Animals

We obtained adult hermaphrodites of the self-fertilizing Kryptolebias marmoratus (n = 107; 6–12 months old) from a breeding colony in the Hagen Aqualab at the University of Guelph, Guelph, Ontario, Canada. Our experiments were performed on fish from the isogenic HON11 strain, which originated from the Bay Islands, Utila, Honduras [13]. Although the HON11 strain has been maintained in the laboratory for approximately 25 years [13], adult hermaphrodites produce genetically identical offspring and the resulting isogenic strains remain stable for several generations [14]. We held fish in 120 ml containers under constant conditions (approx. 60 ml, 15‰, 25°C, pH approx. 8, 12 : 12 h light : dark cycle) and fed fish brine shrimp (Artemia sp. nauplii) three times per week. We acclimated fish for 7 days to water of pH 6.7 (approx. 60 ml, 15‰, 22°C) before the start of the experiment because of the lower pH found in natural mangrove swamps [12]. Moreover, conducting experiments at lower pH values ensures a higher proportion of H₂S is present, compared to less-toxic sulfide species (HS⁻ and S²⁻) [15].

(b) . Experimental protocol

(i) . Hydrogen sulfide and visual acuity

To determine visual acuity, we used an optokinetic response (OKR) machine modified from existing designs [16,17]. We transferred fish into a cuvette plugged with mesh, which was then placed in a stationary water-filled beaker within the rotating drum of the OKR machine (10.5 r.p.m.). We gradually decreased the size of black and white gratings on the rotating drum to identify the smallest grating that elicited a visual response [16].

We tested visual acuity in fish exposed to one of three H₂S concentrations: approximately 100, 200 or 400 µM of H₂S (pH 6.7, 15‰; n = 9–12 per [H₂S]), prepared using Na₂S · 9H₂O. Since K. marmoratus leap out of the water (emerse) to escape H₂S when concentrations approach 200 µM [8,12], we chose H₂S concentrations at, above and below this threshold. We first tested the visual acuity of fish in H₂S-free water after a 10 min acclimation to the OKR machine (control). We then replaced the water in the beaker with H₂S-rich water and waited for 10 min before repeating the OKR test. Finally, we allowed fish to recover in H₂S-free water for 10 min, before assessing visual acuity a third time. A 10 min acclimation was used to provide enough time for fish to adjust to their surroundings while minimizing the time for H₂S oxidation/volatilization. We repeated the experiment at 400 µM of H₂S in a separate subset of fish but used a 1 h recovery period (n = 13). We recorded all responses through a dissection microscope (WILD Heerbrugg C.B.S. 155) with a cell phone camera and adaptor (Carson Universal Optical Smartphone Adaptor). Water samples were taken from each treatment before and after the visual acuity test to verify H₂S concentrations [15,18] (electronic supplementary material, table S1).

(ii) . Cytochrome c oxidase activity

To determine if COX activity was impacted by H₂S, we exposed new groups of fish to either approximately 100, 200 or 400 µM of H₂S for 10 min, and then euthanized fish in ice water before quickly dissecting the eyes (n = 6–9 per treatment group). We also assessed the COX activity of control fish (n = 10), as well as fish allowed to recover from approximately 100, approximately 200 µM of H₂S for 10 min and from approximately 400 µM of H₂S for either 10 min or 1 h (n = 7–8 per recovery group). We homogenized tissues on ice in 200 µl of homogenization buffer (20 mmol l⁻¹ Hepes, 1 mmol l⁻¹ sodium EDTA and 0.1% Triton X-100) using a hypodermic needle and then centrifuged tissue homogenates at 20 000g (4 min) to remove cell debris. We measured COX activity in the supernatant (µM g wet mass⁻¹ min⁻¹) as previously described [8,19].

(iii) . Calculations and statistics

Visual acuity (cycles degree⁻¹) was calculated for each fish using the following equation:

$$\mathrm{visual}\hspace{0.17em}\mathrm{acuity}=\hspace{0.17em}\frac{1}{2{\tan }^{-1}(h/2a)}$$

where a is the distance between the fish's eye and the grating (6.5 cm), and h is the length of the smallest cycle (the combined length of one black and one white line on the grating) to elicit a visual response.

We assessed data for normality and homogeneity of variance using Shapiro–Wilk and Bartlett's tests, respectively, and log-transformed when necessary. We used repeated-measures ANOVAs, followed by Tukey's post hoc tests, to compare the visual acuity of fish between their control, treatment and recovery tests. We analysed the COX data with a one-way ANOVA, followed by a Dunnett's many-to-one comparison test to determine which treatment and recovery groups differed from the control (significant at α < 0.05). We used RStudio (v. 1.1.463 [20]) with R (v. 3.6.1 [21]) for all statistical analyses and Prism (v. 8.0.2) for graphing.

3. Results

(a) . Visual acuity

Visual acuity was impaired at the higher H₂S concentrations. At approximately 100 µM H₂S, there were no significant changes in visual acuity between control, treatment and recovery tests (ANOVA, p = 0.62; figure 1a). Exposure to approximately 200 µM of H₂S significantly reduced visual acuity relative to control (ANOVA, p = 0.002; Tukey, p = 0.006), but visual acuity was restored to control levels following recovery in H₂S-free water (Tukey, p = 0.99; figure 1b). At approximately 400 µM of H₂S, visual acuity also declined relative to the control (ANOVA, p = 0.003; Tukey; p = 0.002), but vision did not fully recover after 10 min in H₂S-free water, as visual acuity during recovery was not significantly different from control (Tukey, p = 0.15) or treatment levels (Tukey; p = 0.12; figure 1c). A longer recovery (1 h), however, restored visual acuity to control levels (ANOVA, p < 0.0001; Tukey, p < 0.67; figure 1d).

Figure 1.

The visual acuity (cycles degree⁻¹) of adult mangrove rivulus (Kryptolebias marmoratus) during consecutive control, treatment and recovery OKR tests. Fish were tested at either (a) low (approx. 100 µM), (b) medium (approx. 200 µM) or (c,d) high (approx. 400 µM) H₂S concentrations. Different letters indicate significance (α < 0.05). Means ± s.e.m. (n = 9–13 per group).

(b) . Cytochrome c oxidase activity

COX activity in the eye decreased in response to the higher H₂S concentrations (ANOVA, p < 0.0001). COX activity was significantly lower in fish exposed to approximately 200 µM of H₂S (Dunnett's, p = 0.02) and approximately 400 µM of H₂S (Dunnett's, p = 0.005; figure 2) relative to the control. At the highest concentration, COX activity did not recover after 10 min (Dunnett's; p = 0.005), but was fully recovered after 1 h (Dunnett's, p = 0.53; figure 2).

Figure 2.

COX activity (µM g wet mass⁻¹ min⁻¹) in the eye tissue of adult mangrove rivulus (Kryptolebias marmoratus). Fish were tested under control conditions (0 µM of H₂S; n = 10) and compared to treatment and recovery groups of low (approx. 100 µM; treatment n = 6, recovery n = 8), medium (approx. 200 µM; treatment n = 8, recovery n =8) and high (approx. 400 µM; treatment n = 9, recovery n = 7, 1 h recovery n = 7) H₂S concentrations. Means ± s.e.m. Red asterisks indicate groups that are statistically different from control (α < 0.05).

4. Discussion

Our results indicate that H₂S concentrations greater than or equal to 200 µM impaired visual acuity and COX activity in the eye, consistent with our hypothesis. Fish appeared to have a graded response to increasing H₂S concentrations, with no impairment of visual acuity or COX activity at lower H₂S concentrations, but impairment at higher concentrations, requiring longer recovery times at the highest concentrations (approx. 400 µM of H₂S). It is possible that the laboratory-reared fish used in our study may respond differently to H₂S than wild fish, which are routinely exposed to H₂S in their natural environment. However, we previously demonstrated that both laboratory-reared and wild K. marmoratus emerse at similar H₂S concentrations (approx. 200 µM [8,12]), suggesting that H₂S responses may not change considerably even after years in captivity. Emersion at approximately 200 µM by laboratory-reared and wild fish may be a strategy to avoid visual impairment. Interestingly, in a previous study, we found wild K. marmoratus occupying mangrove pools where H₂S concentrations exceeded 1000 µM [12]. Therefore, there may be circumstances when K. marmoratus remain in H₂S-rich water for prolonged periods of time (e.g. to avoid terrestrial predators), resulting in negative impacts on the visual system.

We found that K. marmoratus recover relatively quickly from moderate H₂S exposure. Recovery in H₂S-free water depends on both diffusive loss of H₂S [22], as well as detoxification mechanisms [6,23]. H₂S is detoxified in tissues (e.g. liver, spleen, kidney and gills) and blood by the oxidation of H₂S by thiosulfate, catalysed by quinone oxidoreductase [6,7]. At high H₂S concentrations, we found that recovery took up to 1 h, presumably because a larger H₂S load required more time to diffuse from the tissues and/or be detoxified. Some H₂S-tolerant fishes express an H₂S-resistant COX1 isoform [10,23]; however, this does not appear to be the case in K. marmoratus [8], making them more susceptible to the toxic effects of H₂S.

Environmental H₂S may directly damage eye tissue. In humans (and other mammals), gaseous H₂S causes edema of the cornea, eye irritation and potentially blindness [24]. In a preliminary investigation, we did not observe any profound physical damage to the eye of K. marmoratus at approximately 400 µM of H₂S (electronic supplementary material, figure S1). Interestingly, the H₂S-tolerant cave-dwelling Poecilia mexicana has reduced eyes, possibly because they inhabit H₂S-rich and low light environments [25]. By contrast, K. marmoratus have relatively large eyes for their body size and possess a rete mirabile (electronic supplementary material, figure S1) which would facilitate O₂ delivery to the eye. Furthermore, K. marmoratus do not form sulphaemoglobin when exposed to elevated H₂S, which would prevent a reduction in the blood O₂ carrying capacity [8]. Thus, K. marmoratus may have evolved mechanisms to minimize visual impairment while living in a relatively toxic environment—an interesting avenue for future investigation.

Ethics

Experiments were approved by the University of Guelph Animal Care Committee (AUP 3891).

Data accessibility

The datasets supporting this article have been uploaded as part of the electronic supplementary material.

Authors' contributions

C.A.A., G.S.R. and P.A.W. designed the experiments. C.A.A. and G.S.R. carried out the work. C.A.A. and P.A.W. wrote the draft manuscript. C.A.A., G.S.R. and P.A.W. edited the manuscript. All authors approve the final version and agree to be held accountable for the work performed.

Competing interests

I/We declare I/we have no competing interests.

Funding

This work was supported by the National Sciences and Engineering Research Council of Canada (NSERC) Discovery grants to P.A.W. and a NSERC graduate scholarship to G.S.R.