Air exposure moderates ocean acidification effects during embryonic development of intertidally spawning fish

Abiotic parameters

The temperature regime experienced by the embryos was purposefully natural and therefore varied between the three air exposure treatments. The subtidal treatment, where embryos were continuously submerged in water, remained around 9.5 °C for the duration of the experiment, while the intertidal treatments experienced dips in temperature during outside air exposure down to 2.5 °C and 0.8 °C, for the low and high intertidal respectively (Fig. 3A). Accumulated thermal units (ATU; days × temperature post collection until hatch) for each air exposure treatment were 79.6, 75.4 and 65.3 for subtidal, low intertidal and high intertidal, respectively. Despite differences in thermal regime, peak hatch was on the same day (March 14, 2021) for all air exposure and CO₂ treatments, estimated at 11 dpf.

The pH levels in the tanks were measured hourly and were stable over the course of the embryonic incubation period, with no overlap between treatments, although there was some overlap between individual jars. Control treatment was consistently around a pH of 8, the medium treatment had a pH of 7.4 and the high CO₂ treatment had a mean pH of 7.1 (Table 1). After hatch, when the larvae were transferred to the jars, circulation and gas exchange between jars and tank were not as high and CO₂ accumulated in the jars over time, leading to pH levels deviating from tank pH levels (Fig. 3B). Although oxygen levels remained high (7–9 mg/L), the pH dropped from a mean 8–7.6 in the control on two occasions, and was brought back up with a partial water exchange from the incubation tank water. The pH in the medium and high CO₂ treatments were not as affected (Fig. 3B), however, final water chemistry measurements after completion of the experiment (2 days post water exchange) revealed much higher CO₂ levels in all treatments (Table 1: day 15).

Effect of air exposure and CO₂ treatment during embryonic development

Neither embryonic survival nor growth were significantly affected by treatment in our experiment. Percent daily embryonic mortality was low and not significantly affected by CO₂ treatment or air exposure (CO₂: p = 0.088, F₂ = 2.45; Tide: p = 0.11, F₂ = 2.19; CO₂*Tide: p = 0.18, F₂ = 1.59) . Egg diameter at 6 dpf was also not significantly affected by treatment (CO₂: p = 0.38, X² (2, N = 30) = 1.92; Tide: p = 0.83, X² (2, N = 30) = 0.33; CO₂*Tide: p = 0.08, X² (2, N = 30) = 8.25). Metabolic rate, as indicated by embryonic heart rate, was significantly affected by air exposure at 6 dpf (p < 0.001, X² (2, N = 12) = 17.38), but not CO₂ (p = 0.09, X² (2, N = 12) = 4.74 or CO₂*Tide (p = 0.15, X² (2, N = 12) = 6.66. Interestingly, in the medium CO₂ treatment, embryonic heart rate was significantly lower in the high intertidal treatment compared to the low intertidal and subtidal treatment (p = 0.027, X² (2, N = 12) = 8.6), while in the high CO₂ treatment, heart rate was significantly increased by 10% in the low intertidal compared to the subtidal and high intertidal regime (p = 0.007, X² (2, N = 12) = 12.0) (Fig. 4A, Tables 2 and 3).

Overall hatch success was very high in all treatments, and ANOVA did not show a significant affect of CO₂ (p = 0.203; F₂ = 1.75), air exposure (p = 0.668, F₂ = 0.41) nor the interaction (p = 0.768, F₄ = 0.45). Effect size analysis showed a significant positive effect of tidal exposure in the ambient CO₂ treatment, with 14% higher hatch rate in the high intertidal treatment compared to the subtidal treatment (Table 3, Fig. 4B).

Figure 4C shows the effect of air exposure and CO₂ on larval size at hatch. Both CO₂ and tidal exposure were significant (CO₂: p = 0.007, X² (2, N = 30) = 9.8; Tide: X² (2, N = 30) = 40.6), with length negatively correlated with air exposure. The greatest effect was seen in the medium CO₂ treatment, where short air exposure lead to 2% smaller larvae in the low intertidal treatment compared to no air exposure, and long air exposure in the high intertidal treatment lead to 6% smaller larvae compared to no air exposure in the subtidal treatment (Table 3). Length at hatch was also significantly affected by CO₂ treatment, with smaller larvae hatching in the medium and high CO₂ treatment with prolonged air exposure compared to control CO₂ conditions. For larvae reared in high CO₂ conditions, only long air exposure in the high intertidal treatment had a significant effect on size at hatch with larvae 5% smaller than those with no air exposure in the subtidal treatment (for a summary of statistics, see Tables 2 and 3).

Carry-over effects on the larval phase

The greatest effect of air exposure was the reduction in cumulative larval mortality from hatch to yolk-sac depletion at 6 dph (Fig. 4D). Larval mortality was significantly affected by tidal exposure (p < 0.001, F₂ = 11.21), but not CO₂ (p = 0.198, F₂ = 1.78) nor the interaction (p = 0.869, F₄ = 0.31). Significant differences in mortality were found in all air exposed treatments relative to the subtidal, fully immersed treatment, with a 70%—80% reduction in larval mortality with prolonged air exposure in the high intertidal across CO₂ treatments (Table 3).

Analyses of larval morphology, including length and height, wet and dry weight, yolk sac area and depletion rate, spinal and cranial morphology, and eye diameter revealed no significant effects of CO₂ and air exposure, either alone or in combination with a pca. The daily growth rate computed for individual larvae from length at 5 dph relative to the mean length at hatch for the respective replicate, was significantly affected by air exposure (p = 0.002, X² (2, N = 30) = 12.46), with growth rate increasing with increasing air exposure in the medium CO₂ treatment (Tables 2 and 3). Interestingly, yolk sac depletion rate was not significantly affected by CO₂ (p = 0.75, X² (2, 30) = 0.57) or air exposure (p = 0.26, X² (2, 30) = 2.71), and there was no significant interaction (p = 0.92, X² (4, 30) = 0.96). Cranial abnormalities were found in very few the larvae and these were not correlated with treatment or other morphometric components in a pca.

Larval dry weight: wet weight was significantly affected by CO₂ (p = 0.003, F₂ = 1.66) and the interaction between CO₂ and tide was marginally significant (p = 0.060, F₄ = 2.26). In a linear mixed model relating wet weight to length, CO₂ and tidal exposure, length and CO₂ were significant (length: p < 0.001, X² (1, N = 10) = 139.3; CO₂: p = 0.001, X² (2, N = 10) = 13.08) and the interaction of the three parameters was marginally significant (p = 0.08, X² (4, N = 10) = 8.12). Figure 5 shows the relationship between wet weight and length for different CO₂ and tidal treatments. The control treatment had a higher slope with moderate air exposure in the low intertidal treatment, indicating improved condition in this treatment compared to the subtidal treatment (Fig. 5). Prolonged air exposure in the high intertidal, on the other hand, significantly increased the slope and therefore improved condition in the medium and high CO₂ treatments. Note that the slopes of weight-length relationships in the low intertidal control treatment and the high intertidal medium CO₂ treatment are the same, indicating that prolonged air exposure improved larval condition in the medium treatment up to the levels of the control. In contrast, in the high intertidal treatment condition was improved with air exposure but not up to levels seen in the high CO₂ treatment (Fig. 5).

Source: Ecology - nature.com