In zebrafish (Danio rerio), six distinct Na+-K+-ATPase (NKA) alpha1-subunit genes have been identified, and four of them, zatp1a1a.1, zatp1a1a.2, zatp1a1a.4, and zatp1a1a.5, are expressed in embryonic skin where different types of ionocytes appear. The present study attempted to test a hypothesis of whether these NKA alpha1 paralogues are specifically expressed and function in respective ionocytes. Double fluorescence in situ hybridization analysis demonstrated the specific expression of zatp1a1a.1, zatp1a1a.2, and zatp1a1a.5 in NKA-rich (NaR) cells, Na+-Cl- cotransporter (NCC)-expressing cells, and H+-ATPase-rich (HR) cells, respectively, based on the colocalization of the three NKA alpha1 genes with marker genes of the respective ionocytes (epithelial Ca2+ channel in NaR cells; NCC in NCC cells; and H+-ATPase and Na+/H+ exchanger 3b in HR cells). The mRNA expression (by real-time PCR) of zatp1a1a.1, zatp1a1a.2, and zatp1a1a.5 were, respectively, upregulated by low-Ca2+, low-Cl-, and low-Na+ freshwater, which had previously been reported to stimulate uptake functions of Ca2+, Cl-, and Na+. However, zatp1a1a.4 was not colocalized with any of the three types of ionocytes, nor did its mRNA respond to the ambient ions examined. Taken together, zATP1a1a.1, zATP1a1a.2, and zATP1a1a.5 may provide driving force for Na+-coupled cotransporter activity specifically in NaR, NCC, and HR cells, respectively.

Understanding Na+ uptake mechanisms in vertebrates has been a research priority since vertebrate ancestors were thought to originate from hyperosmotic marine habitats to the hypoosmotic freshwater system. Given the evolutionary success of osmoregulator teleosts, these freshwater conquerors from the marine habitats are reasonably considered to develop the traits of absorbing Na+ from the Na+-poor circumstances for ionic homeostasis. However, in teleosts, the loss of epithelial Na+ channel (ENaC) has long been a mystery and an issue under debate in the evolution of vertebrates. In this study, we evaluate the idea that energetic efficiency in teleosts may have been improved by selection for ENaC loss and an evolved energy-saving alternative, the Na+/H+ exchangers (NHE3)-mediated Na+ uptake/NH4+ excretion machinery. The present study approaches this question from the lamprey, a pioneer invader of freshwater habitats, initially developed ENaC-mediated Na+ uptake driven by energy-consuming apical H+-ATPase (VHA) in the gills, similar to amphibian skin and external gills. Later, teleosts may have intensified ammonotelism to generate larger NH4+ outward gradients that facilitate NHE3-mediated Na+ uptake against an unfavorable Na+ gradient in freshwater without consuming additional ATP. Therefore, this study provides a fresh starting point for expanding our understanding of vertebrate ion regulation and environmental adaptation within the framework of the energy constraint concept.

Background: Cephalopods have evolved strong acid-base regulatory abilities to cope with CO 2 induced pH fluctuations in their extracellular compartments to protect gas transport via highly pH sensitive hemocyanins. To date, the mechanistic basis of branchial acid-base regulation in cephalopods is still poorly understood, and associated energetic limitations may represent a critical factor in high power squids during prolonged exposure to seawater acidification. Results: The present work used adult squid Sepioteuthis lessoniana to investigate the effects of short-term (few hours) to medium-term (up to 168 h) seawater acidification on pelagic squids. Routine metabolic rates, NH 4 + excretion, extracellular acid-base balance were monitored during exposure to control (pH 8.1) and acidified conditions of pH 7.7 and 7.3 along a period of 168 h. Metabolic rates were significantly depressed by 40% after exposure to pH 7.3 conditions for 168 h. Animals fully restored extracellular pH accompanied by an increase in blood HCO 3 − levels within 20 hours. This compensation reaction was accompanied by increased transcript abundance of branchial acid-base transporters including V-type H +-ATPase (VHA), Rhesus protein (RhP), Na + /HCO 3 − cotransporter (NBC) and cytosolic carbonic anhydrase (CAc). Immunocytochemistry demonstrated the sub-cellular localization of Na + /K +-ATPase (NKA), VHA in basolateral and Na + /H +-exchanger 3 (NHE3) and RhP in apical membranes of the ion-transporting branchial epithelium. Branchial VHA and RhP responded with increased mRNA and protein levels in response to acidified conditions indicating the importance of active NH 4 + transport to mediate acid-base balance in cephalopods. Conclusion: The present work demonstrated that cephalopods have a well developed branchial acid-base regulatory machinery. However, pelagic squids that evolved a lifestyle at the edge of energetic limits are probably more sensitive to prolonged exposure to acidified conditions compared to their more sluggish relatives including cuttlefish and octopods.

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Exposure of ectothermic vertebrates to fluctuated temperatures accelerates mitochondrial respiration and has been shown to increase the formation of mitochondrial reactive oxygen species (ROS), such as O2−, H2O2, and O2−·. Excess ROS production by intensively respiring mitochondria is held responsible for cellular damage. The present study was to test whether a protective oxidative‐stress pathway would be initiated in fish central nervous system upon cold stress.Effects of acute cold exposure (from 28 °C to 18 °C) on brain oxidative stress parameters were investigated in zebrafish (Danio rerio). Concentrations of cellular protein carbonyl groups (biomarkers of oxidative stress) were significantly increased after 1‐hour cold exposure. Increased levels of anti‐oxidative stress parameters, catalase (CAT) and superoxide dismutase (SOD) were observed at 1‐ and 6‐hour cold exposure, respectively. In spite of maintained anti‐oxidant capacity, the increment status of cellular oxidized protein accumulation was still observed. A neuronal defense pathway against ROS, peroxisome proliferator‐activated receptor (PPAR), acts with genes encoding uncoupling proteins (UCPs) was further monitored. Real‐time PCR analysis indicated that ppar‐1αb increased was transcriptionally regulated the expression of zucp2 and zucp2l. Mitochondrial biogenesis activation, mild uncoupling, and reduction of free radicals may result intracellular pO2 levels drop. Moreover, these protective mechanisms in fish brain have been found to cause hypoxia‐induced factor (HIF) stabilization that facilitates an increase in energy supply.

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Osmotic and ionic homeostasis in the body fluid is important for vertebrates including teleost fish, and this is mainly achieved in gill/skin ionocytes in fish. Chloride is the most abundant anion in animal tissue and plasma and participates in many physiological processes. Comparing with the regulation mechanisms of other ions that have been studied in details in zebrafish, the chloride ion regulatory pathways are less understood. In the present study, we cloned out 11 members of clc chloride channels in zebrafish. Several clc members were found to be expressed in the gills of zebrafish by RT‐PCR; however, only the mRNA of clc2b was localized by in situ hybridization in the zebrafish embryonic yolk sac and adult fish gill with an ionocyte pattern. Combination of clc2b in situ hybridization and zebrafish sodium chloride cotransporter‐like 2 (zNCC‐like 2) immunocytochemistry indicated that 2 transporters are co‐expressed on the same ionocyte. Furthermore, comparison of chloride content of clc2b morpholino knockdown and wild‐type zebrafish embryos showed significant difference. Altogether, the data suggested that clc2b plays an important role in zebrafish chloride regulation.