Ion regulation at gills precedes gas exchange and the origin of vertebrates.

Gas exchange and ion regulation at gills have key roles in the evolution of vertebrates1-4. Gills are hypothesized to have first acquired these important homeostatic functions from the skin in stem vertebrates, facilitating the evolution of larger, more-active modes of life2,3,5. However, this hypothesis lacks functional support in relevant taxa. Here we characterize the function of gills and skin in a vertebrate (lamprey ammocoete; Entosphenus tridentatus), a cephalochordate (amphioxus; Branchiostoma floridae) and a hemichordate (acorn worm; Saccoglossus kowalevskii) with the presumed burrowing, filter-feeding traits of vertebrate ancestors6-9. We provide functional support for a vertebrate origin of gas exchange at the gills with increasing body size and activity, as direct measurements in vivo reveal that gills are the dominant site of gas exchange only in ammocoetes, and only with increasing body size or challenges to oxygen supply and demand. Conversely, gills of all three taxa are implicated in ion regulation. Ammocoete gills are responsible for all ion flux at all body sizes, whereas molecular markers for ion regulation are higher in the gills than in the skin of amphioxus and acorn worms. This suggests that ion regulation at gills has an earlier origin than gas exchange that is unrelated to vertebrate size and activity-perhaps at the very inception of pharyngeal pores in stem deuterostomes.

A solution to Nature's haemoglobin knockout: a plasma-accessible carbonic anhydrase catalyses CO2 excretion in Antarctic icefish gills.

In all vertebrates studied to date, CO2 excretion depends on the enzyme carbonic anhydrase (CA) that catalyses the rapid conversion of HCO3- to CO2 at the gas-exchange organs. The largest pool of CA is present within red blood cells (RBCs) and, in some vertebrates, plasma-accessible CA (paCA) isoforms participate in CO2 excretion. However, teleost fishes typically do not have paCA at the gills and CO2 excretion is reliant entirely on RBC CA - a strategy that is not possible in icefishes. As the result of a natural knockout, Antarctic icefishes (Channichthyidae) are the only known vertebrates that do not express haemoglobin (Hb) as adults, and largely lack RBCs in the circulation (haematocrit <1%). Previous work has indicated the presence of high levels of membrane-bound CA activity in the gills of icefishes, but without determining its cellular orientation. Thus, we hypothesised that icefishes express a membrane-bound CA isoform at the gill that is accessible to the blood plasma. The CA distribution was compared in the gills of two closely related notothenioid species, one with Hb and RBCs (Notothenia rossii) and one without (Champsocephalus gunnari). Molecular, biochemical and immunohistochemical markers indicate high levels of a Ca4 isoform in the gills of the icefish (but not the red-blooded N. rossii), in a plasma-accessible location that is consistent with a role in CO2 excretion. Thus, in the absence of RBC CA, the icefish gill could exclusively provide the catalytic activity necessary for CO2 excretion - a pathway that is unlike that of any other vertebrate.

Water pH limits extracellular but not intracellular pH compensation in the CO2-tolerant freshwater fish Pangasianodon hypophthalmus.

Preferentially regulating intracellular pH (pHi) confers exceptional CO2 tolerance on fish, but is often associated with reductions in extracellular pH (pHe) compensation. It is unknown whether these reductions are due to intrinsically lower capacities for pHe compensation, hypercarbia-induced reductions in water pH or other factors. To test how water pH affects capacities and strategies for pH compensation, we exposed the CO2-tolerant fish Pangasianodon hypophthalmus to 3 kPa PCO2  for 20 h at an ecologically relevant water pH of 4.5 or 5.8. Brain, heart and liver pHi was preferentially regulated in both treatments. However, blood pHe compensation was severely reduced at water pH 4.5 but not 5.8. This suggests that low water pH limits acute pHe but not pHi compensation in fishes preferentially regulating pHi Hypercarbia-induced reductions in water pH might therefore underlie the unexplained reductions to pHe compensation in fishes preferentially regulating pHi, and may increase selection for preferential pHi regulation.

The origins of gas exchange and ion regulation in fish gills: evidence from structure and function.

Gill function in gas exchange and ion regulation has played key roles in the evolution of fishes. In this review, we summarize data from the fields of palaeontology, developmental biology and comparative physiology for when and how the gills first acquired these functions. Data from across disciplines strongly supports a stem vertebrate origin for gas exchange structures and function at the gills with the emergence of larger, more active fishes. However, the recent discovery of putative ionocytes in extant cephalochordates and hemichordates suggests that ion regulation at gills might have originated much earlier than gas exchange, perhaps in the ciliated pharyngeal arches in the last common ancestor of deuterostomes. We hypothesize that the ancestral form of ion regulation served a filter-feeding function in the ciliated pharyngeal arches, and was later coopted in vertebrates to regulate extracellular ion and acid-base balance. We propose that future research should explore ionocyte homology and function across extant deuterostomes to test this hypothesis and others in order to determine the ancestral origins of ion regulation in fish gills.