This thesis examined the ionoregulatory physiology of freshwater-adapted mummichog (𝘍𝘶𝘯𝘥𝘶𝘭𝘶𝘴 𝘩𝘦𝘵𝘦𝘳𝘰𝘤𝘭𝘪𝘵𝘶𝘴) 𝘪𝘯 𝘷𝘪𝘷𝘰 and its interaction with acid-base regulation. Under control conditions (water [NaCl] ≈ 0.7-1 .0 mmol· L⁻¹, [Ca²⁺] ≈ 0.1 or 1.0 mmol· L⁻¹), Na⁺ turnover was vigorous with a positive Na⁺ balance maintained, whereas unidirectional Cl⁻ influx was virtually zero resulting in a slight negative Cl⁻ balance. Michaelis-Menten analysis revealed a low affinity, high capacity Na⁺ uptake mechanism that was independent of both Na⁺ efflux and ammonia excretion. Cl⁻ uptake started at higher water [NaCl] levels ( > 2 mmol • L⁻¹) but did not saturate within the freshwater [NaCl] range, indicating a completely different uptake mechanism, independent from Na⁺ influx. Using both internal and environmental manipulation, Na⁺ uptake was found not to be coupled to ammonia excretion. Instead, a link with acid excretion (Na⁺ /H⁺ exchange or Na⁺ channel/H⁺ -ATPase coupling) remains possible but could not be confirmed. There was no evidence for the presence of a Cl⁻/HC0₃⁻exchange mechanism in the gills. However, mummichog were capable of differentially manipulating Na⁺ and Cl⁻ efflux components as an additional response to an internal acid-base disturbance. This ability and the suggested Na⁺ uptake/acid excretion coupling indicate that mummichog resemble other freshwater fish in that an iono/acid-base relationship exists. In these studies, the use of the Strong lon Difference Theory as a means of assessing acid-base balance through the measurement of differential Na⁺ and Cl⁻ fluxes proved to be acceptable and practical alternative to the measurement of acid-base fluxes by traditional titration methodology. Finally, whole-body Ca²⁺ uptake was investigated using a recently developed technique for small fish. Ca²⁺ uptake by the mummichog involves a carrier-mediated step as revealed by saturation of uptake as external [Ca²⁺] increased. Inhibition of uptake by external La³⁺ but not Mg²⁺ suggested that apical Ca²⁺ channels are involved in the uptake process but are not voltage-gated. Chronic exposure to low Ca²⁺ water resulted in a stimulated Ca²⁺ uptake, most likely in response to depletion of internal Ca²⁺ levels whereas chronic exposure to high Ca²⁺ did not elicit any changes in uptake. This thesis revealed that the freshwater-adapted mummichog does share certain ionoregulatory qualities with other freshwater fish but at the same time possesses unique characteristics which may reflect its euryhaline nature / Thesis / Master of Science (MS)

For organisms relying on an aerobic metabolism, a constant oxygen (O₂) supply must be available to energy demanding tissues. In this thesis. the effects of hypoxia exposure and altered ionoregulatory demands on O₂ uptake of the larval zebrafish (Danio rerio) were evaluated. In Chapter 2, it was hypothesized that a pre-exposure to hypoxia would alter the O₂ uptake capacity of 4- and 7-days post-fertilisation (dpf) larvae through a modified vasculature system. Additionally, using a genetic knockout line, the role of Hif-1α in regulating cutaneous O₂ flux (JO₂) was tested. It was predicted that hypoxia-exposed larvae would display a higher JO₂ across the body due to a hypoxic, acclimatory response, explained by an increased vascularity and supported by an increased whole-body O₂ consumption (ṀO₂) and decreased critical O₂ tension (Pcrit). Consequently, this response was expected to be negated in the Hif1aa⁻/⁻ab⁻/⁻ larvae. Ultimately, JO₂ measured using the scanning micro-optrode technique (SMOT) remained unchanged between WT and Hif1aa⁻/⁻ab⁻/⁻ and normoxia- and hypoxia-exposed larvae, a finding which was supported by an unchanged vascularity across all treatments. The results from this chapter suggest that changes in hypoxia performance mediated by Hif-1α are unrelated to cutaneous JO₂ and vascularity. In Chapter 3, the aerobic costs of ion transport in 4 dpf larval zebrafish was assessed. We hypothesized that changes in rates of Na⁺ uptake evoked by acidic or low Na⁺ rearing would result in changes in ṀO₂ and/or JO₂, measured at the ionocyte-expressing yolk sac epithelium using SMOT. Ultimately, it was found that the measured JO₂ and ṀO₂ did not correlate with the corresponding Na⁺ uptake rate triggered by the acidic and low Na⁺ rearing environment. 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 for a low aerobic cost for ion regulation in zebrafish larvae.

Largely through the use ofin vitro preparations there is now a generally accepted theory for ion transport for the seawater (SW) gill. However, to date there is no generally accepted freshwater (FW) model for the mechanisms of NaCl transport in the teleost gill. By using an Ussing chamber approach with the opercular epithelia of Fundulus heteroclitus and Oreochromis niloticus, and the urinary bladder of Oncorhynchus mykiss, all acclimated to FW, we hoped to establish one as a possible model for the study of FW ion regulation. FW Fundulus opercular epithelia displayed a serosal negative transepithelial potential (Vₜ) of-43.9 mV, transepithelial conductance (Gₜ) of 1.94 mS·cm⁻², and active transport of Cl⁻ from the mucosal FW against a strong electrochemical gradient. Na⁺ movement was dominated by passive diffusion. The opercular epithelia of Fundulus adapted to 10% SW exhibited properties similar to SW Fundulus by actively extruding Cl⁻ while Na⁺ moved passively into the mucosal 10% SW. With FW bathing the mucosal surface, FW Oreochromis opercular epithelia displayed a serosal positive Vₜ of +8.0 mV, Gₜ of 1.78 mS·cm⁻², and active reabsorption of Na⁺, Cl⁻ and Ca²⁺ against large electrical and/or chemical gradients. The FW Oreochromis opercular epithelia is the only FW in vitro preparation to date that exhibits active absorption (albeit at small absolute rates) of both Na⁺ and Cl⁻. FW 0. mykiss urinary bladders mounted in vitro under symmetrical saline conditions exhibited a transepithelial conductance (Gₜ) of-9.15 mS·cm⁻² and electroneutral active absorption of Na⁺ and Cl⁻ from the mucosal urine side. The transport of Na⁺ and Cl⁻ was a partially coupled process whereby removal of Na⁺ from the mucosal saline decreased Cl⁻ absorption by a 56% and removal of Cl⁻ inhibited Na⁺ absorption by 69%. However, active net absorption of both ions persisted when the counter-ion was replaced with a non-permeant ion. Under more realistic conditions with artificial urine bathing the mucosal surface, Vₜ increased to a serosal positive ~+7.6 mV and Gₜ decreased to ~1.47 mS·cm⁻² Unidirectional influx rates of both Na⁺ and Cl⁻ were much lower, but active absorption of both ions still occurred. Replacement of Na⁺ in the mucosal artificial urine caused no change in unidirectional influx of Cl⁻ and vice versa. The mucosal addition of DIDS, amiloride or bumetanide (10⁻⁴M) all had no affect on absorption rates of Na⁺ and/or Cl⁻, under either artificial urine or symmetrical saline conditions. When the mucosal surface was bathed in artificial urine, removal of mucosal Cl⁻· significantly reduced the maximum transport rate (Jₘₐₓ of Na⁺ (6.1 ~2.1 μmol·cm⁻²·h⁻¹) but had no effect on affinity for Na⁺ (Kₘ~27 mM). Similarly, removal of mucosal Na⁺ significantly reduced the Jₘₐₓ for Cl⁻ uptake (11.4 ~2.4 μmol·cm⁻²·h⁻¹) but had no effect on Cl⁻ Kₘ(~37 mM). The anterior portion of the urinary bladder transported Na⁺ and Cl⁻ at a faster rate than the posterior portion under symmetrical saline conditions, but there was no difference in measured Na⁺/K⁺ -ATPase activities between the two portions. In conclusion, opercular epithelia of Fundulus and Oreochromis demonstrated transport properties believed to be typical of FW teleosts; either preparation may eventually prove to be a good working model for studying FW transport mechanisms. The urinary bladder of Oncorhynchus mounted in vitro did not behave characteristically as the teleost gill is thought to function, but this model may be useful in characterizing various other mechanisms of ionic transport. / Thesis / Master of Science (MSc)

Relative to other metals, little is known about lead toxicity in fish. The use of predictive models such as the biotic ligand model (BLM) has been limited, a situation that is at least partially due to the lack of understanding of lead's acute toxic mechanism and characterization of key binding sites involved in this toxicity. Using the rainbow trout as a model species, the acute toxic mechanism for lead was found to be ionoregulatory disruption. While having no apparent respiratory or acid/base effects, Pb exposure resulted in significant ionoregulatory impacts that affected Ca2+ homeostasis, as well as Na+ and Cl- balance. Active Ca2+ uptake by the gills obeyed typical Michaelis-Menten kinetics, and Pb interacted in a competitive fashion with the uptake process. Exposure to increasing waterborne Pb concentrations resulted in significant increases in Km value while Jmax showed little or no change. A slower, non-competitive interaction occurred after prolonged Pb-exposure, evidenced by a significant reduction of high-affinity Ca2+ -ATPase activity that correlated well with branchial Pb accumulation. Conversely, calcium had a protective effect against branchial Pb accumulation, this relationship being predominately competitive in nature. Voltage-independent calcium channel blockers La3+, Cd, and Zn significantly reduced gill Pb burden while the voltage-dependent, L-type calcium channel blockers, nifedipine and verapamil, did not, suggesting Pb enters fish by a similar mechanism to that of Ca2+. Stimulated stanniocalcin release by CaCl2 injection also significantly reduced branchial Pb accumulation. Based on the evidence presented in this thesis, it is apparent that acute Pb toxicity occurs by ionoregulatory disruption. It is likely that Pb shares a similar uptake pathway as that for Ca2+ and that resulting accumulation results in disruption of Ca2+ influx as well as Na+ and Cl- balance. This study has provided data essential to the characterization of key binding sites involved in Pb toxicity, and ultimately, validates the development and application of predictive models such as the BLM. / Thesis / Master of Science (MSc)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The purpose of this study was to verify the effect of dietary salt (NaCl) supplementation, water hardness and pH on growth, survival and ionoregulation of silver catfish (Rhamdia quelen) juveniles. In the first experiment, juveniles were fed with diets supplemented with 0.0; 0.5; 1.0 e 2.0 % NaCl and exposed to pH 5.5, 7.0 and 9.0 for 35 days. In the second study, fish were maintained for 30 days in three different pH (5.5; 7.0 and 9.0) and four water hardness (30, 60, 120, and 180 mg L-1 CaCO3). The third experiment investigated the effects of the 6.0-8.0 pH range at low water hardness (0, 25 and 50 mg L-1 CaCO3) for 32 days. In the experiments 1 and 2, fish samples were collected at different moments for analyses of Na+, K+ and Cl- net ion fluxes. In all three studies, growth and survival were analyzed. The water utilized was previously adjusted to the appropriate pH and water hardness using NaOH or H2SO4 0.5 M and CaCl2.2H2O, respectively. In the first experiment, fish fed with diet without NaCl supplementation and exposed to pH 7.0 presented significantly higher weight, length, SGR and biomass per tank than those exposed to pH 5.5, and the increase of dietary NaCl protected against the impact of acidic water. Dietary salt supplementation contributed to decrease the osmoregulatory disturbances in the juveniles exposed to acidic or basic pH. In the second study, exposure of juveniles to alkaline or acidic water did not affect survival, but acidic water reduced growth. And, finally, in the third study, juveniles exposed to pH 7.0 and 8.0 at zero water hardness showed significantly higher mortality than those kept at pH 6.0. Survival and growth of juveniles exposed to 25 and 50 mg L-1 CaCO3 was not affected in the 6.0-8.0 pH range. Therefore, the best water hardness for silver catfish juveniles growth and osmoregulation is 30-60 mg L-1 CaCO3 and at low water hardness (next zero) pH must be reduced. It can be concluded that the interaction of dietary salt, pH and water hardness are very important to silver catfish rearing, because they change growth and ionoregulation in this species. / A proposta deste estudo foi verificar o efeito da adição de sal na dieta, dureza da água e pH no crescimento, sobrevivência e ionorregulação de juvenis de jundiá (Rhamdia quelen). No primeiro experimento, juvenis foram alimentados com dietas suplementadas com 0,0; 0,5; 1,0 e 2,0 % NaCl e expostos aos pH 5,5; 7,0 e 9,0 por 35 dias. No segundo estudo, peixes foram mantidos por 30 dias em três pH (5,5; 7,0 e 9,0) e quatro durezas da água (30, 60, 120 e 180 mg L-1 CaCO3). O terceiro experimento investigou os efeitos do pH dentro da faixa de 6.0-8.0 em baixa dureza da água (0, 25 e 50 mg L-1 CaCO3) por 32 dias. Nos experimentos 1 e 2, exemplares foram coletados em diferentes momentos para análise dos fluxos iônicos líquidos de Na+, K+ e Cl. Em todos os três estudos, crescimento e sobrevivência foram analisados. A água utilizada foi previamente ajustada para o pH e dureza da água apropriados usando NaOH ou H2SO4 0,5 M e CaCl2.2H2O, respectivamente. No primeiro experimento, peixes alimentados com dietas sem adição de NaCl e expostos a pH 7,0 apresentaram peso, comprimento, SGR e biomassa por tanque significativamente maiores que aqueles mantidos em pH 5,5, e o aumento de NaCl na dieta protegeu contra o impacto da água ácida. A inclusão de sal na dieta reduziu os distúrbios osmorregulatórios nos juvenis expostos a pH ácido ou básico. No segundo trabalho, juvenis expostos a águas alcalinas ou ácidas não tiveram sua sobrevivência afetada, mas o crescimento foi reduzido em água ácida. E finalmente, no terceiro estudo, juvenis expostos a pH 7,0 e 8,0 em dureza zero da água apresentaram mortalidade significativamente maior que aqueles mantidos em pH 6.0. Nos juvenis expostos a 25 e 50 mg L-1 CaCO3 a sobrevivência e o crescimento não foram afetados na faixa de pH 6,0-8,0. Portanto, a melhor dureza da água para crescimento e osmorregulação de juvenis de jundiá se encontra entre 30-60 mg L-1 CaCO3, e em dureza baixa (próxima de zero) deve-se reduzir o pH da água. Pode-se concluir que a interação de parâmetros como sal na dieta, pH e dureza da água são deveras importantes no cultivo do jundiá, uma vez que alteram o crescimento e a ionorregulação desta espécie.

Insects maintain blood (haemolymph) Ca2+ concentrations within a narrow range in order to support the health of internal tissues and organs. The Malpighian (renal) tubules play a primary role in haemolymph Ca2+ homeostasis by sequestering excess Ca2+ within calcified biomineral deposits (Ca-rich granules) often located within type I (principal) tubule cells. Using the classic Ramsay assay, the scanning ion-selective microelectrode technique (SIET), and modifications of these two electrophysiological techniques, this thesis begins to unravel the sites and mechanisms of Ca2+ transport by the Malpighian tubules isolated from eight insects, representing seven orders. A segment-specific pattern of Ca2+ flux was observed along the length of the Malpighian tubules isolated from D. melanogaster, A. aegypti and A. domesticus and was uniform along the length in the remaining species. The majority (≥ 90%) of Ca2+ entering the tubule cells is sequestered within intracellular calcium stores in Ca2+-transporting segments of D. melanogaster and A. domesticus tubules, consistent with the presence of Ca-rich storage granules in these tubule segments. In addition, this thesis provides the first measurements of basolateral Ca2+ flux across single principal and secondary tubule cells of T. ni, where Ca2+ uptake occurs only across principal cells. Perhaps the most important finding of this thesis is that increasing fluid secretion through manipulation of intracellular levels of cAMP or Ca2+ in isolated tubules of A. domesticus had opposite effects on tubule Ca2+ transport. The adenylyl cyclase-cAMP-PKA pathway promotes Ca2+ sequestration whereas both 5-hydroxytryptamine and thapsigargin inhibited sequestration. In contrast, tubules of the remaining species were generally insensitive to cAMP or thapsigargin and v rates of tubule Ca2+ transport were often very low. The presence of Ca-rich granules in the cells of the midgut in several of the species with low rates of tubule Ca2+ transport provide evidence for a putative role of the midgut in haemolymph Ca2+ homeostasis. Taken together, these results suggest that the principal cells of the Malpighian tubules contribute to haemolymph calcium homeostasis through neuroendocrine regulated sequestration of excess Ca2+ during periods of high dietary calcium intake. Sequestration of dietary Ca2+ by the midgut may reduce Ca2+ entry into the haemolymph and therefore Ca2+ sequestration by the Malpighian tubules need not be so rapid. Finally, reversible tubule Ca2+ transport may allow internal reserves of Ca2+ (Ca-rich granules) to be returned to the haemolymph allowing insects to survive prolong periods of Ca2+ deficiency (i.e. overwintering). / Thesis / Doctor of Philosophy (PhD) / This thesis contributes to our understanding of how insects regulate the calcium content of their blood (haemolymph). Using electrophysiological techniques with improved spatial resolution (from millimeters to micrometers) this thesis sought to determine the sites, mechanisms and regulation of Ca2+ transport by insect Malpighian (renal) tubules in order to gain insights into the role of Ca-rich granules (similar to those identified in early stages of human kidney stone formation i.e. nephrolithiasis) within these tissues. Using eight insect species this thesis demonstrates that the Malpighian tubules act as dynamic Ca2+ stores that appear to be under neuroendocrine control: actively taking up Ca2+ through calcium entry channels, where the majority (≥ 90%) of excess haemolymph Ca2+ is sequestered within intracellular stores (Ca-rich granules) during period of excess dietary calcium and passively releasing Ca2+ back to the haemolymph during periods of metamorphosis or calcium deficiency (i.e. overwintering).