Loss-of-function technologies, such as morpholino-and RNAimediated gene knockdown, and TALEN-and CRISPR/Cas9mediated gene knockout, are widely used to investigate gene function and its physiological significance. Here, we provide a general overview of the various knockdown and knockout technologies commonly used in comparative physiology and discuss the merits and drawbacks of these technologies with a particular focus on research conducted in zebrafish. Despite their widespread use, there is an ongoing debate surrounding the use of knockdown versus knockout approaches and their potential off-target effects. This debate is primarily fueled by the observations that, in some studies, knockout mutants exhibit phenotypes different from those observed in response to knockdown using morpholinos or RNAi. We discuss the current debate and focus on the discrepancies between knockdown and knockout phenotypes, providing literature and primary data to show that the different phenotypes are not necessarily a direct result of the off-target effects of the knockdown agents used. Nevertheless, given the recent evidence of some knockdown phenotypes being recapitulated in knockout mutants lacking the morpholino or RNAi target, we stress that results of knockdown experiments need to be interpreted with caution. We ultimately argue that knockdown experiments should not be discontinued if proper control experiments are performed, and that with careful interpretation, knockdown approaches remain useful to complement the limitations of knockout studies (e.g. lethality of knockout and compensatory responses).

Hemoglobin (Hb) multiplicity is common in fish, yet despite its ubiquitous nature, the functional significance is unclear. Here we explore the hypothesis that Hb multiplicity plays a role in hypoxia tolerance using the red drum (Sciaenops ocellatus). Red drum is an economically and ecologically important species native to coastal regions and estuaries of the Gulf of Mexico – habitats that routinely experience pronounced hypoxic events. Using a transcriptomic approach, we demonstrate that red drum red blood cells express 7 and 5 Hbα and Hbβ isoforms, respectively. Phylogenetic analysis grouped these isoforms into distinct isoHb clades, and provided evidence of lineage specific expression of particular isoHbs. In normoxia, three isoHbs predominated (Hbα-3.1, -3.2, and Hbβ-3.1). A three-week hypoxia acclimation (48 mmHg) resulted in significant up-regulation of Hbα-2, Hbα-3.2, and Hbβ-3.1, effectively switching the predominantly expressed isoforms. Changes in subunit expression were correlated with a decrease in non-stripped hemolysate P50. Similarly, hypoxia acclimation resulted in a 20% reduction in whole animal critical oxygen threshold (Pcrit). Hypoxia acclimation was not associated with changes in gill morphology, hematocrit, or relative ventricular mass. Overall, these data provide support for the hypothesis that Hb isoform switching can provide a physiological benefit to counteract environmental stress in fishes.

Mass-specific oxygen consumption rate, i.e. standard metabolic rate (Rs ) and critical oxygen tension (Pcrit ) of red drum Sciaenops ocellatus were measured and scaled over a 2500-fold range in mass (MF ; 0·26-686 g). Rs conformed to well established models (Rs  = 3·73·91 MF (-0·21) ; r(2)  = 0·86) while Pcrit increased over the size range (Pcrit  = 3·15 log10 MF  + 16·19; r(2)  = 0·44). This relationship may be ecologically advantageous as it would allow smaller S. ocellatus to better utilize hypoxic zones as habitat and refuge from predators.

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.

Fish increase ventilation during hypoxia, a reflex termed the hypoxic ventilatory response (HVR). The HVR is an effective mechanism to increase O 2 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 (Pw O2) at which this decline occurs is expected to be near the critical Pw O2 (P crit), the Pw O2 at which O 2 consumption begins to decline. Our results indicate that in zebrafish (Danio rerio), the relationship between peak HVR and P crit is dependent on developmental stage. Peak ventilation occurred at Pw O2 values higher than P crit in larvae, but at a Pw O2 significantly lower than P crit 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 Pw O2 below P crit .