Hemoglobin enhances oxygen uptake in larval zebrafish (Danio rerio) but only under conditions of extreme hypoxia

Abstract

SUMMARYThe role of hemoglobin (Hb) in O 2 uptake by zebrafish larvae ranging in age from 5 to 42 days postfertilization was assessed under conditions of normoxia, moderate hypoxia and extreme hypoxia. This was achieved by exposing larvae with and without functional Hb to continuously declining oxygen levels (P O2 ) in closed-system respirometers. Exposure to 5% CO for 2-4 h was used to render Hb effectively non-functional in terms of its ability to transport O 2 . Routine metabolic rate (rM O2 ), critical dissolved oxygen level (P c ) and residual oxygen level (P r ) were determined and used, respectively, as indicators of response in normoxia, moderate hypoxia and extreme hypoxia. rM O2 was defined as the average rate of O 2 uptake before O 2 became limiting (i.e. at high P O2 s). P c is the P O2 at which rM O2 first becomes O 2 -limited and P r is the P O2 below which larvae are no longer able to extract O 2 from the ambient medium. CO poisoning had no significant impact on rM O2 or P c at any age, indicating that the lack of functional Hb does not impair routine O 2 usage in normoxia or at moderate levels of hypoxia [down to at least 25-50 torr (1 torrϷ0.133 kPa), depending on age]. P r , however, was significantly lower overall for control larvae (6.7±1.1 torr; mean ± 95%CI) than for COpoisoned larvae (11.2±2.1 torr). It would appear that the presence of functional Hb allows zebrafish larvae to extract O 2 from water down to lower P O2 s under conditions of extreme hypoxia. This is the first documented (as opposed to inferred) benefit of Hb in developing zebrafish. However, given the relatively small magnitude of the effect it is unclear if this benefit on its own is sufficient to balance the costs associated with Hb production and maintenance.

Carbonic anhydrase expression and CO2 excretion during early development in zebrafishDanio rerio

“…; Fig.2) were comparable with those reported in other studies carried out on zebrafish of similar developmental stage and at similar temperatures (Pelster and Burggren, 1996;Barrionuevo and Burggren, 1999;Bagatto et al, 2001;Grillitsch et al, 2005;Rombough and Drader, 2009). By contrast, the present study appears to be the first to report measurements of CO 2 excretion during early development in a fish.…”

“…By contrast, several studies using a variety of experimental approaches have demonstrated that O 2 uptake in developing zebrafish is not dependent on haemoglobin O 2 transport (via RBCs) until at least 12-14d.p.f. (Pelster and Burggren, 1996;Weinstein et al, 1996;Jacob et al, 2002;Rombough and Drader, 2009; see review by Rombough, 2004) even though erythrogenesis is initiated around 15h.p.f. and RBCs containing haemoglobin are present in the circulation by 24h.p.f.…”

“…The involvement of CA in CO 2 excretion in larval zebrafish may imply a role for the RBC and could help to explain the appearance of RBCs early in development. In particular, the selective advantage of early haemoglobin synthesis has not been obvious given that it is not needed for O 2 transport (Rombough and Drader, 2009). Based on the findings of the present study, i.e.…”

Aquatic surface respiration and swimming behaviour in adult and developing zebrafish exposed to hypoxia

“…During early developmental stages, the gills may not be fully developed and so zebrafish larvae use both cutaneous and gill respiration to satisfy metabolic needs for O 2 (Rombough, 2002). In addition, a model has predicted that zebrafish can survive by simple diffusion in air-saturated water without the need of a circulatory system until 42 dpf (Rombough and Drader, 2009). ASR may then become important as a survival strategy in larvae at O 2 tensions closer to anoxia, where reduced activity no longer provides any benefit and an escape response is elicited.…”

Vitreoscilla Hemoglobin (VHb) Overexpression Increases Hypoxia Tolerance in Zebrafish (Danio rerio)

“…Because early life stage (ELS) is one of the most sensitive life stages to toxicity during the life cycle, ELS tests with fish species are commonly used as routine toxicity tests in risk assessment (OECD 1998;Roex et al 2002). D. rerio was recommended as a model fish species for short-term embryo-larval toxicity testing (OECD 1998), and there are many studies investigating the effects of hypoxia on zebrafish embryo or larvae (Padilla and Roth 2001;Ton et al 2003;Rombough and Drader 2009;Steele et al 2009;Van Rooijen et al 2009;Yaqoob et al 2009). We found that the 7-day-old F 3 vhb transgenic larvae may be suitable for study of the hypoxia tolerance traits.…”