Evaluating the physiological significance of hypoxic hyperventilation in larval zebrafish (Danio rerio)

Abstract

In water-breathing fishes, the hypoxic ventilatory response (HVR) represents an increase in water flow over the gills during exposure to lowered ambient O 2 levels. The HVR is a critical defense mechanism that serves to delay the negative consequences of hypoxia on aerobic respiration. However, the physiological significance of the HVR in larval fishes is unclear as they do not have a fully developed gill and rely primarily on cutaneous gas transfer. Using larval zebrafish (4, 7, 10 and 15 days post-fertilization; dpf), we examined HVR under three levels of hypoxia (25, 45 and 60 mmHg). The larvae exhibited widely different HVRs as a function of developmental age and level of the hypoxia. Yet, critical O 2 tensions (P crit) remained constant (30-34 mmHg) over the same period of development. Micro-optrode O 2 sensors were used to measure a significant decrease in buccal cavity water O 2 tensions in 4 and 7 dpf larvae compared with the water they inspired, demonstrating significant extraction of O 2 from the buccal cavity. To assess the physiological significance of the HVR, ventilatory water flow was prevented in larvae at 4 and 7 dpf by embedding their heads in agar. An increase in P crit was observed in larvae at 7 dpf but not 4 dpf, suggesting that buccal ventilation is important for O 2 extraction by 7 dpf. Combined, these data indicate that branchial/buccal gas transfer plays a significant role in O 2 uptake during hypoxia, and supports a physiological benefit of the HVR in early life stages of zebrafish.

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

“…S1). Thus the HVR is not necessary to maintain O 2 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.…”

“…S1). Moreover, at 7 dpf (unlike at 4 dpf ), preventing hypoxic hyperventilation in zebrafish impedes O 2 uptake (Pan et al, 2019). In older larvae, the HVR, coupled with cutaneous respiration, becomes an important mechanism to maintain ṀO 2 , and a shift of maximal ventilatory effort closer to that of P crit would be beneficial to O 2 uptake.…”

“…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 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 ṀO 2 as the severity of hypoxia increases, but despite the benefits of the HVR, ṀO 2 eventually declines in severe hypoxia at a Pw O2 termed the critical O 2 tension (P crit ).…”

“…In zebrafish (Danio rerio), cutaneous diffusion is the dominant mechanism of O 2 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 O 2 uptake (Pan et al, 2019). Therefore, in zebrafish, both branchial and cutaneous respiration contribute to O 2 uptake during larval stages, with the proportional contribution of each shifting over developmental time.…”