Respirometry and cutaneous oxygen flux measurements reveal a negligible aerobic cost of ion regulation in larval zebrafish (Danio rerio).

Fishes living in fresh water counter the passive loss of salts by actively absorbing ions through specialized cells termed ionocytes. Ionocytes contain ATP-dependent transporters and are enriched with mitochondria; therefore ionic regulation is an energy-consuming process. The purpose of this study was to assess the aerobic costs of ion transport in larval zebrafish (Danio rerio). We hypothesized that changes in rates of Na+ uptake evoked by acidic or low Na+ rearing conditions would result in corresponding changes in whole-body oxygen consumption (MO2 ) and/or cutaneous oxygen flux (JO2 ), measured at the ionocyte-expressing yolk sac epithelium using the scanning micro-optrode technique (SMOT). Larvae at 4 days post-fertilization (dpf) that were reared under low pH (pH 4) conditions exhibited a higher rate of Na+ uptake compared with fish reared under control conditions (pH 7.6), yet they displayed a lower MO2  and no difference in cutaneous JO2 Despite a higher Na+ uptake capacity in larvae reared under low Na+ conditions, there were no differences in MO2  and JO2  at 4 dpf. Furthermore, although Na+ uptake was nearly abolished in 2 dpf larvae lacking ionocytes after morpholino knockdown of the ionocyte proliferation regulating transcription factor foxi3a, MO2 and JO2  were unaffected. Finally, laser ablation of ionocytes did not affect cutaneous JO2 Thus, we conclude that the aerobic costs of ion uptake by ionocytes in larval zebrafish, at least in the case of Na+, are below detection using whole-body respirometry or cutaneous SMOT scans, providing evidence that ion regulation in zebrafish larvae incurs a low aerobic cost.

Does hypoxia-inducible factor 1α play a role in regulating cutaneous oxygen flux in larval zebrafish (Danio rerio)?

Previous studies have demonstrated that hypoxia tolerance is improved in zebrafish (Danio rerio) larvae after prior exposure to lowered ambient O2 levels. Such improved hypoxia performance was attributed in part, to increased levels of hypoxia-inducible factor 1α (Hif-1α) exerting downstream effects on various physiological processes including promotion of trunk skin angiogenesis. Since O2 uptake ([Formula: see text]) in larvae is facilitated largely by O2 diffusion across the skin, enhanced cutaneous vascularization is expected to enhance [Formula: see text] during hypoxia and thus contribute to improved hypoxia tolerance. In this study, we used the scanning micro-optrode technique together with quantification of cutaneous vascularity in wild types (WT) and Hif-1α knockouts (hif1aa-/-ab-/-) to test the hypothesis that improved hypoxia tolerance after hypoxia acclimation in larvae at 4 or 7 days post-fertilization (dpf) was associated with Hif-1α-dependent increases in skin vascularity and regional cutaneous O2 fluxes (JO2). Hypoxia tolerance, as determined by measurements of critical PO2 (Pcrit), was unaltered by hypoxia pre-exposure in larvae at 4 dpf and there were no significant differences in Pcrit between WT and hif1aa-/-ab-/- larvae at this developmental stage. However, at 7 dpf there was a significant effect of genotype with WT larvae showing a lower Pcrit than hif1aa-/-ab-/- larvae, an effect that was being driven by a reduced Pcrit in the WT larvae after hypoxia pre-exposure (19.2 ± 1.9 mmHg) compared to hif1aa-/-ab-/- fish (35.5 ± 3.5 mmHg). Regardless of genotype, pre-exposure status or developmental age, JO2 decreased along the body in the anterior-to-posterior direction. Neither hypoxia pre-exposure nor genotype affected JO2 at any region along the body. The lack of any effect of hypoxia pre-exposure or genotype on JO2 was consistent with the lack of any effect on skin vascularity as measured in Tg(fli1:EGFP)yl transgenic larvae. Thus, the decreased hypoxia performance (increased Pcrit) at 7 dpf in the hif1aa-/-ab-/- larvae did not appear to be reliant on changes in trunk vascularity or cutaneous O2 diffusion.