Relationships between the peak hypoxic ventilatory response and critical O₂ tension in larval and adult zebrafish (Danio rerio)

Environmental disturbances, particularly hypoxia, can compromise branchial gas transfer and thus rapid physiological adjustments are initiated to minimize the impact on O₂ uptake (Ṁ_O2; Perry and Wood, 1989). Most teleost species increase ventilation volume through a change in ventilation frequency (f_V) and/or amplitude (reviewed by Perry et al., 2009), referred to as the hypoxic ventilatory response (HVR). The HVR helps to maintain arterial P_O2 in the face of decreasing water P_O2 (Pw_O2; Perry et al., 2009) and, typically, the magnitude of the HVR is dependent on the severity of hypoxia (e.g. Sundin et al., 1999; Vulesevic et al., 2006; Pan et al., 2019). The HVR is an important factor delaying an inevitable decrease in Ṁ_O2 as the severity of hypoxia increases, but despite the benefits of the HVR, Ṁ_O2 eventually declines in severe hypoxia at a Pw_O2 termed the critical O₂ tension (P_crit). Similarly, in many fish species, ventilation volume increases with the severity of hypoxia to a peak, after which ventilatory effort declines with further decreases in Pw_O2 (Rantin et al., 1992; Cerezo and Garcia Garcia, 2004; Scott et al., 2008; Monteiro et al., 2013). This decline in the HVR in severe hypoxia may be a result of diminishing benefits of the HVR (Perry et al., 2009). The metabolic cost of ventilation is high, and even at rest may account for 10% of routine Ṁ_O2 (Cameron and Cech, 1970; Jones and Schwarzfeld, 1974; Randall and Daxboeck, 1984) owing to the high density and viscosity of water combined with high ventilation convection requirements (Perry and Wood, 1989; Gilmour, 1997). In severe hypoxia, the increase in ventilation volume incurs a metabolic cost at a time when O₂ is limited, leading to a possible mismatch between a reduced capacity for ATP production and increased metabolic demand of respiratory tissues. Therefore, any benefit of increased O₂ uptake from the HVR may not be sufficient to sustain the cost of ventilation, resulting in a decline in HVR during hypoxia. However, to date no study has explicitly determined the cause of the decline in HVR and it is possible that other limitations may play a role. Based on a correlation of data gleaned from the literature, it was suggested that peak ventilation occurs near the P_crit (Perry et al., 2009), and this observation led Wood (2018) to suggest that fish ‘abandon’ hyperventilation at or near the P_crit. However, the relationship has not been tested experimentally by collecting ventilation and P_crit data from the same individual.

Moreover, we predict that this relationship will change over development. In zebrafish (Danio rerio), cutaneous diffusion is the dominant mechanism of O₂ uptake in larvae until the gills become the primary site of gas transfer at around 15 days post-fertilization (dpf; Rombough, 2002; Rombough, 2004). Despite the apparently limited respiratory role of the gills during early developmental stages, larval zebrafish begin to hyperventilate in response to hypoxia as early as 3 dpf (Jonz and Nurse, 2005), and by 7 dpf, preventing hyperventilation impairs O₂ uptake (Pan et al., 2019). Therefore, in zebrafish, both branchial and cutaneous respiration contribute to O₂ uptake during larval stages, with the proportional contribution of each shifting over developmental time. During stages when cutaneous respiration is dominant, maintaining hyperventilation over a wide Pw_O2 range may be less important than in adult fish. Thus, we predict peak ventilation will occur at higher Pw_O2 than P_crit. For adult zebrafish, we predict that the Pw_O2 corresponding to peak ventilation during progressive hypoxia will be near P_crit but unlike the assertion of Wood (2018), we expect that hyperventilation will continue as Pw_O2 falls below P_crit. In addition to characterizing the relationship between P_crit and peak ventilatory effort in adult and larval zebrafish across developmental time, we updated the survey of the literature to include data on peak HVR and P_crit of 11 more species not included in the analysis of Perry et al. (2009). Maintaining peak ventilatory effort is metabolically costly, particularly when O₂ is limited, and discerning peak ventilation patterns in relation to Ṁ_O2 during progressive hypoxia may provide an important indicator as to when the metabolic cost of maintaining HVR outweighs the benefit of increased O₂ uptake.

Species for which peak ventilation (typically reported as ventilation volume with the exception of a few studies that measured water flow) during hypoxia and P_crit were known were used in a correlation analysis of the Pw_O2 of peak ventilation and P_crit. With a few exceptions, peak ventilation and P_crit were obtained from different batches of fish within a single study. In four studies, one on sharpsnout sea bream (Diplodus puntazzo) (Cerezo and Garcia Garcia, 2004), two on Nile tilapia (Oreochromis niloticus) and one on Amazonian oscar (Astronotus ocellatus), peak ventilation and P_crit were measured simultaneously in response to progressive hypoxia. For rainbow trout (Oncorhynchus mykiss), peak ventilation and P_crit were obtained from two separate sources.

Adult zebrafish, Danio rerio (F. Hamilton 1822), were held in 10 l polycarbonate tanks in a recirculating aquatic system (Aquatic Habitats, Apopka, FL, USA) at the University of Ottawa aquatic care facility. Fish were kept under a 14 h:10 h light:dark cycle in 28°C dechloraminated city of Ottawa tap water and were fed to satiation with GEMMA 300 fish feed (Skretting USA, Westbrook, ME, USA) twice daily. Standard breeding protocols (Westerfield, 2000) were followed to obtain embryos during controlled breeding events. The night before breeding, a male zebrafish was separated by a divider from two female zebrafish in a 2 l breeding tank. The following morning, the water was changed and the divider was removed, allowing the fish to breed. Embryos were collected and reared in 50 ml Petri dishes (40 embryos per dish) containing dechloraminated city of Ottawa tap water and 0.05% Methylene Blue maintained at 28°C. Water in the Petri dishes was replaced daily. At 5 dpf, the larvae were transferred to static 2 l tanks and water was changed in the tanks every second day. At this stage, the larvae begin to feed exogenously and fish were fed daily to satiation with GEMMA Micro 75 fish feed (Skretting USA). The larvae were raised to 7, 10 and 15 dpf. All procedures for animal use and experimentation were carried out in compliance with the University of Ottawa Animal Care and Veterinary Service guidelines under protocol BL-226 and adhered to the recommendations for animal use provided by the Canadian Council for Animal Care.

Simultaneous measurements of Ṁ_O2 and f_V in response to declining Pw_O2 were recorded in adult zebrafish and larvae at 4, 7, 10 and 15 dpf. Adult zebrafish were placed into 15.6 ml glass respirometers fitted with O₂ sensor spots (horizontal mini chamber system; Loligo Systems, Viborg, Denmark) and allowed to recover overnight. The respirometers were flushed continuously with 28°C water from a 20 l recirculating tank gassed with air. At the beginning of the trial, the flush pump to the glass respirometer was turned off while the recirculating pump remained on to provide mixing to ensure stable Pw_O2 readings. Pw_O2 was monitored continuously in the closed system respirometer using AutoResp (Loligo Systems). Pw_O2 fell as fish consumed the O₂ in the respirometer and the experiment was terminated when the Pw_O2 levels plateaued. Each individual fish was video recorded for the duration of the Ṁ_O2 trial using an iPhone SE camera and f_V data were extracted from the videos by manual counting as described below. The mass of each fish was determined using an analytical balance.

A larva was placed into an 80 μl respirometry well, fitted with an O₂ sensor spot (24-well glass microplate; Loligo Systems) and situated on an O₂ sensor reader (SDR SensorDish Reader, PreSens, Regensburg, Germany). Both the microplate and the fluorescence sensor were placed under a dissecting microscope (stereo trinocular microscope, AmScope, Irvine, CA, USA) focused on the well containing the larva. The experiment was conducted in a temperature-controlled room maintained at 28°C. The well was sealed with adhesive tape (AB0580, ThermoFisher Scientific, Mississauga, ON, Canada) at the beginning of the trial and Pw_O2 levels were monitored using MicroResp (Loligo Systems) until the experiment was terminated upon the plateauing of P_O2 levels. For the duration of the trial, the fish was video recorded using an iPhone SE camera mounted on the dissecting microscope. The mass of 4, 7, 10 and 15 dpf zebrafish larvae was determined on a separate batch of fish using the protocol of Pan et al. (2019).

The Ṁ_O2 was calculated over sequential 3 min intervals using the slope of the relationship of Pw_O2 versus time, standardized for fish mass and respirometer volume. Water O₂ concentration was calculated using the solubility coefficient of O₂ in freshwater at 28°C (Boutilier et al., 1984). The Ṁ_O2 was plotted as a function of Pw_O2 and an inflection point representing P_crit was calculated for each fish using the broken-stick (or segmented) regression approach (Yeager and Ultsch, 1989) and REGRESS software (www.wfu.edu/~mudayja/sofware/o2.exe).

In both adults and larvae, f_V was quantified by counting either buccal or opercular movements depending on the orientation of the fish in the chamber or well and the visibility of the mouth and/or operculum. We focused on f_V as an index of ventilation volume because adult zebrafish increase f_V, not breathing amplitude, during hypoxia (Vulesevic et al., 2006), and there is no established method to measure amplitude in larval zebrafish. Average f_V was determined for the first minute of each 3 min bin used to calculate Ṁ_O2. The f_V was plotted against Pw_O2 and an inflection point, termed the ‘f_V inflection point’, was determined using the broken-stick (or segmented) regression approach, the same technique used to calculate P_crit. Although it is straightforward to determine the Pw_O2 of peak ventilation, this value may not be fully representative of the response because often there is a range of Pw_O2 values over which ventilation plateaus near maximum values. We found that the variance around the mean P_crit for larvae and adults was on average approximately 17%, and we chose this value to represent the range of ventilation near peak value, which we termed the ‘zone of maximal ventilation’.

Tg(fli1:eGFP) larvae that express enhanced green fluorescent protein (GFP) in the vasculature under the control of the fli1 promoter were raised to 4, 7, 10 and 15 dpf and fixed overnight by immersion in 4% paraformaldehyde in phosphate-buffered saline at 4°C. Larvae were mounted in 1% low melt agarose (BioShop, Burlington, ON, Canada) on a depression slide (VWR, Mississauga, ON, Canada) and images were acquired using a Nikon A1R MP confocal microscope with Apo ×25/1.10 NA water objective and NIS-elements software (Nikon Instruments Inc., Melville, NY, USA).

Statistical analyses were performed in R (https://www.R-project.org/). The linear association between P_crit and Pw_O2 at peak ventilation was determined using Pearson's product moment correlation coefficient. Whether P_crit was significantly different from the f_V inflection at each larval stage and in adult zebrafish was tested using a two-tailed Student's t-test. An ANOVA in the car package (Fox and Weisberg, 2011) was used to determine whether the difference between P_crit and f_V inflection varied with developmental stage, and Tukey's post hoc test was performed on P_crit−f_V inflection (Δ). All data were tested for normality using the Shapiro–Wilk test and equal variance using Bartlett's test. Data that failed normality or equal variance were log transformed. Significance was set at P<0.05.

The goal of this study was to characterize the relationship between P_crit and peak HVR in larval and adult zebrafish. During hypoxia, ventilation increases as the severity of hypoxia increases, and subsequently falls when Pw_O2 drops to a severely low tension that is species specific (Rantin et al., 1992; Cerezo and Garcia Garcia, 2004; Scott et al., 2008; Monteiro et al., 2013). Perry et al. (2009) proposed that the decline in ventilation occurs around P_crit, the Pw_O2 at which aerobic metabolism is compromised and Ṁ_O2 begins to decrease.

Focusing on 21 fish species for which P_crit and ventilation volume during hypoxia are known (for adult fish), a significant positive correlation was found between P_crit and Pw_O2 at peak ventilation (r=0.81, P<0.01; Fig. 1), indicating that in species with a lower P_crit, peak ventilation also occurred at a lower Pw_O2. A similar survey of the literature on fewer species also obtained a significant correlation between peak HVR and P_crit (Perry et al., 2009). A correlation between peak HVR and P_crit is not surprising given that ventilatory effort is metabolically costly and the effective contribution of ventilation during hypoxia is diminished at P_crit, as evidenced by a fall in Ṁ_O2 (Perry et al., 2009). However, when a line of identity was plotted, most species fell below the line (Fig. 1), indicating that peak ventilation was achieved at a Pw_O2 lower than P_crit, and in some species, like the pacu (Piaractus mesopotamicus), peak ventilation occurred at a Pw_O2 far below P_crit (approximately 20 mmHg lower; Rantin et al., 1998). Thus, despite the apparent significant metabolic cost, in some species, the HVR appears to be maintained even when Pw_O2 falls below P_crit. Thus, the conclusion of Wood (2018) that fish often ‘abandon hyperventilation’ at P_crit does not appear to be supported by existing data presented in Fig. 1.

In adult zebrafish, maximal ventilatory effort occurred at a Pw_O2 that was significantly lower than P_crit (Fig. 2 and Fig. 3A), similar to patterns observed in species such as the spangled perch (Leiopotherapon unicolor; Gehrke and Fielder, 1988), the pacu (Rantin et al., 1998) and the jeju (Hoplerythrinus unitaeniatus; Oliveira et al., 2004) (see Fig. 1). Peak ventilatory effort, quantified either as f_V inflection point or zone of maximal ventilation, occurred around 10 mmHg, well below P_crit (19.9±0.8 mmHg) (Fig. 2 and Fig. 3A). Adult zebrafish are known to have high haemoglobin O₂ affinity (P₅₀=4.4 mmHg; Cadiz et al., 2019), indicating that at the Pw_O2 of maximal HVR, haemoglobin O₂ saturation may have remained near 100%. It is possible that continued hyperventilation at Pw_O2 values below P_crit helps to maintain arterial P_O2, bolstering Ṁ_O2.

In young (<7 dpf) larvae, peak ventilation occurred at a Pw_O2 higher than P_crit but as larvae aged, ventilation peaked at Pw_O2 closer to P_crit (Fig. 2 and Fig. 3). In 4 dpf larvae, the f_V inflection point and zone of maximal ventilation were significantly above P_crit (Figs 2 and 3A), indicating that the HVR was decreasing even as Ṁ_O2 remained constant. At 4 dpf, zebrafish primarily rely on cutaneous respiration (Rombough, 2002; Rombough, 2004) and blood vessels are just beginning to form in the pharyngeal arches region, as can be observed in the image collected using the Tg(fli1:eGFP) line (Fig. 2; Fig. S1). Thus the HVR is not necessary to maintain O₂ uptake at this stage (Jonz and Nurse, 2005; Pan et al., 2019), and a decrease in maximal ventilation at Pw_O2 well above P_crit may be effective in conserving limited metabolic energy. There was a left shift in both the f_V inflection point and zone of maximal ventilation in 7 and 10 dpf larvae, moving them closer to the P_crit (Fig. 2 and Fig. 3A). By 7 dpf, respiratory lamellae begin to form (Jonz and Nurse, 2005), which is apparent in the images collected in the current study as increased vascularization in the buccal cavity (Fig. 2; Fig. S1). Moreover, at 7 dpf (unlike at 4 dpf), preventing hypoxic hyperventilation in zebrafish impedes O₂ uptake (Pan et al., 2019). In older larvae, the HVR, coupled with cutaneous respiration, becomes an important mechanism to maintain Ṁ_O2, and a shift of maximal ventilatory effort closer to that of P_crit would be beneficial to O₂ uptake.

The f_V inflection point and zone of maximal ventilation for 15 dpf occurred at a Pw_O2 above that of P_crit (Fig. 2 and Fig. 3A) and there was no statistical difference between 10 and 15 dpf larvae in P_crit−f_V inflection (Δ) (Fig. 3B). Branchial respiration is thought to dominate in developing zebrafish beginning around 15 dpf (Rombough, 2002). Accordingly, we had expected that the relationship between peak HVR and P_crit at 15 dpf would be similar to that of adult fish, but in contrast, it was more similar to that of younger larvae. In steelhead trout (Oncorhynchus mykiss), P_crit decreases as larvae develop, suggesting an increase in the capacity for O₂ uptake at lower Pw_O2 as development progresses (Rombough, 1988a). In zebrafish larvae, however, P_crit was constant across development to 15 dpf at 32–34 mmHg, whereas in adult fish, P_crit was markedly lower (20 mmHg). Despite the greater reliance on branchial respiration, the full capacity of the adult gill has not yet developed in 15 dpf larvae, likely limiting the capacity to improve O₂ uptake in hypoxia. Regulation of functional gill surface area, ventilation and perfusion is thought to be critical in promoting gas transfer and hypoxia tolerance (Rombough, 1988b). It is possible that these factors cannot be maximized during hypoxia to the same degree in a larval gill as in the adult gill. Aside from changes in convection (e.g. as result of HVR), larval gills show little plasticity compared with adult gills (Sackville and Brauner, 2018), supporting the idea that there may be greater constraints on branchial gas transfer during hypoxia in larvae than in adults. Therefore, it is possible that at 15 dpf, the cost of HVR far exceeds the benefit and the HVR begins to decline at a higher Pw_O2 than P_crit.

By simultaneously measuring Ṁ_O2 and f_V during progressive hypoxia, we evaluated the relationship between peak ventilation frequency and P_crit in developing larvae and adult zebrafish. Peak ventilation occurred at a Pw_O2 significantly higher than P_crit in 4 dpf larvae, but as larvae developed, the zone of peak ventilation shifted to lower Pw_O2 values, closer to P_crit. By adulthood, peak ventilation occurred well below P_crit. The mechanisms that determine the Pw_O2 of maximal HVR are unknown. However, the pattern of changes in the Pw_O2 of peak HVR and P_crit across life history allows us to speculate that a driving factor may be the relationship between the metabolic cost of the HVR versus its benefit. It is likely that in early stage larvae, the metabolic cost of the HVR significantly outweighs its benefit, while the opposite is true in adult fish. However, it is important to consider that the decrease in HVR may not be a result of a shift in balance between metabolic benefit and cost, but rather a result of a different limitation. It is possible that the constraining effects of viscosity on larval fish owing to their small size may produce high demands on ventilatory effort during hypoxia, leading to fatigue of the respiratory muscles. Further research is warranted to determine the underlying cause of the decline in HVR in larval and adult fish.

We thank Christine Archer and other staff at the University of Ottawa aquatic care facility for their help and knowledge of animal husbandry.