Osmoregulatory physiology of pupfish (Cyprinodon spp.) in freshwater

Active uptake of Na+ at the gill by fish is fundamental to their osmoregulation and thus survival in freshwater environments. Studies to date have demonstrated several different mechanisms by which fish can accomplish this important physiological function. This dissertation provides a comparative characterization of Na+ uptake in the coastal pupfish, Cyprinodon variegatus variegatus (Cvv), the closely related Lake Eustis pupfish, C. v. hubbsi (Cvh), and the endangered desert pupfish, C. macularius ( Cm). When acclimated to slightly saline freshwater (7 mM Na +) all three fish use a low affinity Na+/H+ exchanger (NHE) for apical Na+ uptake. Cvv also uses a Na+:K+:2Cl- co-transporter under these conditions, the first time this has been functionally demonstrated in a freshwater fish. When acclimated to 2 mM Na+, all three fish strictly rely on a low affinity NHE for Na+ uptake. Only Cvh, which naturally occurs in eight dilute freshwater lakes in central Florida, was able to acclimate to 0.1 mM Na + and under these conditions switches to a high affinity NHE for Na + uptake. In other fish studied to date that use a high affinity NHE in dilute freshwater, the NHE operates in a metabolon with an Rh glycoprotein (ammonia transporter) that provides H+ for the operation of NHE in this thermodynamically constrained environment. This dissertation confirmed that Cvh does not use an NHE-Rh metabolon, but instead acquires H+ from carbonic anhydrase mediated hydration of CO2 to allow for NHE operation in dilute freshwater. This is the first time this has been demonstrated in a fish exposed to low Na + water. Finally, because Cvh only occurs in eight lakes in central Florida, all of which are suffering from habitat loss and water quality degradation through urban development, this dissertation evaluated whether Cvh should be designated a separate species from Cvv, allowing for greater environmental protection. While there is no clear consensus on the definition of a species, within the regulatory framework of the Endangered Species Act, this dissertation demonstrated heritable physiological differences between Cvv and Cvh, partial pre- and post-zygotic isolation between the two subspecies, and likely monophyletic origin for Cvh, all of which support the designation of Cvh as an evolutionarily significant unit, and likely a separate species.