Fish increase ventilation during hypoxia, a reflex termed the hypoxic ventilatory response (HVR). The HVR is an effective mechanism to increase O2 uptake, but at a high metabolic cost. Therefore, when hypoxia becomes severe enough, ventilation declines, as its benefit is diminished. The water oxygen partial pressure (PwO2) at which this decline occurs is expected to be near the critical PwO2 (Pcrit), the PwO2 at which O2 consumption begins to decline. Our results indicate that in zebrafish (Danio rerio), the relationship between peak HVR and Pcrit is dependent on developmental stage. Peak ventilation occurred at PwO2 values higher than Pcrit in larvae, but at a PwO2 significantly lower than Pcrit in adults. Larval zebrafish use cutaneous respiration to a greater extent than branchial respiration and the cost of sustaining the HVR may outweigh the benefit, whereas adult zebrafish, which rely on branchial respiration, may benefit from using HVR at PwO2 below Pcrit.
Environmental disturbances, particularly hypoxia, can compromise branchial gas transfer and thus rapid physiological adjustments are initiated to minimize the impact on O2 uptake (ṀO2; Perry and Wood, 1989). Most teleost species increase ventilation volume through a change in ventilation frequency (fV) and/or amplitude (reviewed by Perry et al., 2009), referred to as the hypoxic ventilatory response (HVR). The HVR helps to maintain arterial PO2 in the face of decreasing water PO2 (PwO2; 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 ṀO2 as the severity of hypoxia increases, but despite the benefits of the HVR, ṀO2 eventually declines in severe hypoxia at a PwO2 termed the critical O2 tension (Pcrit). 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 PwO2 (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 ṀO2 (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 Pcrit (Perry et al., 2009), and this observation led Wood (2018) to suggest that fish ‘abandon’ hyperventilation at or near the Pcrit. However, the relationship has not been tested experimentally by collecting ventilation and Pcrit 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 PwO2 range may be less important than in adult fish. Thus, we predict peak ventilation will occur at higher PwO2 than Pcrit. For adult zebrafish, we predict that the PwO2 corresponding to peak ventilation during progressive hypoxia will be near Pcrit but unlike the assertion of Wood (2018), we expect that hyperventilation will continue as PwO2 falls below Pcrit. In addition to characterizing the relationship between Pcrit 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 Pcrit 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 ṀO2 during progressive hypoxia may provide an important indicator as to when the metabolic cost of maintaining HVR outweighs the benefit of increased O2 uptake.
MATERIALS AND METHODS
Data mining for peak ventilation and Pcrit in fishes exposed to hypoxia
Species for which peak ventilation (typically reported as ventilation volume with the exception of a few studies that measured water flow) during hypoxia and Pcrit were known were used in a correlation analysis of the PwO2 of peak ventilation and Pcrit. With a few exceptions, peak ventilation and Pcrit 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 Pcrit were measured simultaneously in response to progressive hypoxia. For rainbow trout (Oncorhynchus mykiss), peak ventilation and Pcrit 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.
ṀO2 and fV in adult and larval zebrafish
Simultaneous measurements of ṀO2 and fV in response to declining PwO2 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 PwO2 readings. PwO2 was monitored continuously in the closed system respirometer using AutoResp (Loligo Systems). PwO2 fell as fish consumed the O2 in the respirometer and the experiment was terminated when the PwO2 levels plateaued. Each individual fish was video recorded for the duration of the ṀO2 trial using an iPhone SE camera and fV 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 PwO2 levels were monitored using MicroResp (Loligo Systems) until the experiment was terminated upon the plateauing of PO2 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 ṀO2 was calculated over sequential 3 min intervals using the slope of the relationship of PwO2 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 ṀO2 was plotted as a function of PwO2 and an inflection point representing Pcrit 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, fV 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 fV as an index of ventilation volume because adult zebrafish increase fV, not breathing amplitude, during hypoxia (Vulesevic et al., 2006), and there is no established method to measure amplitude in larval zebrafish. Average fV was determined for the first minute of each 3 min bin used to calculate ṀO2. The fV was plotted against PwO2 and an inflection point, termed the ‘fV inflection point’, was determined using the broken-stick (or segmented) regression approach, the same technique used to calculate Pcrit. Although it is straightforward to determine the PwO2 of peak ventilation, this value may not be fully representative of the response because often there is a range of PwO2 values over which ventilation plateaus near maximum values. We found that the variance around the mean Pcrit 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’.
Confocal imaging of gills in larval zebrafish
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 Pcrit and PwO2 at peak ventilation was determined using Pearson's product moment correlation coefficient. Whether Pcrit was significantly different from the fV 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 Pcrit and fV inflection varied with developmental stage, and Tukey's post hoc test was performed on Pcrit−fV 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.
RESULTS AND DISCUSSION
The goal of this study was to characterize the relationship between Pcrit and peak HVR in larval and adult zebrafish. During hypoxia, ventilation increases as the severity of hypoxia increases, and subsequently falls when PwO2 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 Pcrit, the PwO2 at which aerobic metabolism is compromised and ṀO2 begins to decrease.
Focusing on 21 fish species for which Pcrit and ventilation volume during hypoxia are known (for adult fish), a significant positive correlation was found between Pcrit and PwO2 at peak ventilation (r=0.81, P<0.01; Fig. 1), indicating that in species with a lower Pcrit, peak ventilation also occurred at a lower PwO2. A similar survey of the literature on fewer species also obtained a significant correlation between peak HVR and Pcrit (Perry et al., 2009). A correlation between peak HVR and Pcrit is not surprising given that ventilatory effort is metabolically costly and the effective contribution of ventilation during hypoxia is diminished at Pcrit, as evidenced by a fall in ṀO2 (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 PwO2 lower than Pcrit, and in some species, like the pacu (Piaractus mesopotamicus), peak ventilation occurred at a PwO2 far below Pcrit (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 PwO2 falls below Pcrit. Thus, the conclusion of Wood (2018) that fish often ‘abandon hyperventilation’ at Pcrit does not appear to be supported by existing data presented in Fig. 1.
In adult zebrafish, maximal ventilatory effort occurred at a PwO2 that was significantly lower than Pcrit (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 fV inflection point or zone of maximal ventilation, occurred around 10 mmHg, well below Pcrit (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 PwO2 of maximal HVR, haemoglobin O2 saturation may have remained near 100%. It is possible that continued hyperventilation at PwO2 values below Pcrit helps to maintain arterial PO2, bolstering ṀO2.
In young (<7 dpf) larvae, peak ventilation occurred at a PwO2 higher than Pcrit but as larvae aged, ventilation peaked at PwO2 closer to Pcrit (Fig. 2 and Fig. 3). In 4 dpf larvae, the fV inflection point and zone of maximal ventilation were significantly above Pcrit (Figs 2 and 3A), indicating that the HVR was decreasing even as ṀO2 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 PwO2 well above Pcrit may be effective in conserving limited metabolic energy. There was a left shift in both the fV inflection point and zone of maximal ventilation in 7 and 10 dpf larvae, moving them closer to the Pcrit (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 ṀO2, and a shift of maximal ventilatory effort closer to that of Pcrit would be beneficial to O2 uptake.
The fV inflection point and zone of maximal ventilation for 15 dpf occurred at a PwO2 above that of Pcrit (Fig. 2 and Fig. 3A) and there was no statistical difference between 10 and 15 dpf larvae in Pcrit−fV 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 Pcrit 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), Pcrit decreases as larvae develop, suggesting an increase in the capacity for O2 uptake at lower PwO2 as development progresses (Rombough, 1988a). In zebrafish larvae, however, Pcrit was constant across development to 15 dpf at 32–34 mmHg, whereas in adult fish, Pcrit 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 PwO2 than Pcrit.
By simultaneously measuring ṀO2 and fV during progressive hypoxia, we evaluated the relationship between peak ventilation frequency and Pcrit in developing larvae and adult zebrafish. Peak ventilation occurred at a PwO2 significantly higher than Pcrit in 4 dpf larvae, but as larvae developed, the zone of peak ventilation shifted to lower PwO2 values, closer to Pcrit. By adulthood, peak ventilation occurred well below Pcrit. The mechanisms that determine the PwO2 of maximal HVR are unknown. However, the pattern of changes in the PwO2 of peak HVR and Pcrit 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.
Conceptualization: M.M., Y.K.P., S.F.P.; Methodology: M.M., Y.K.P., S.F.P.; Validation: M.M., Y.K.P., K.M.G., S.F.P.; Formal analysis: M.M., Y.K.P., K.M.G., S.F.P.; Investigation: M.M., Y.K.P.; Resources: S.F.P.; Writing - original draft: M.M.; Writing - review & editing: M.M., Y.K.P., K.M.G., S.F.P.; Visualization: M.M., Y.K.P.; Supervision: S.F.P.; Funding acquisition: S.F.P.
This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant to S.F.P. (G13017) and a Natural Sciences and Engineering Research Council of Canada Post-Doctoral Fellowship to M.M. (PDF-471707-2015).
The authors declare no competing or financial interests.