<p>Tl⁺ is thought to be toxic to cells due to ionic mimicry of K⁺. The aims of this study were two-fold. First, to identify whether K⁺ and Tl⁺ were interacting in isolated guts, whole animals and tissues in <em>Chironomus riparius, </em>and second, to determine the strategies of Tl⁺ tolerance. <em>C. riparius. </em>were very tolerant towards Tl⁺with a 48-hr LC₅₀ of 723 μmol l^-1. The Scanning Ion-selective Technique (SIET) allowed us to identify the caecae, AMG and PMG as the major K⁺-transporting regions of isolated guts. Evidence for an interaction was based on the finding that Tl⁺ was transported in the same directions at these segments (and others), and that 50 μmol l^-1Tl⁺ decreased K⁺ flux at the AMG and PMG. In addition, exposure to Tl⁺ prior to flux measurements had significant effects on net K⁺ transport by the gut. Measurements of Tl⁺ and K⁺ concentrations in the whole animal, gut and hemolymph by Atomic Absorption Spectroscopy (AAS) indicated that Tl⁺ uptake was saturable in the whole animal and gut, and non-saturable in the hemolymph. Together with the SIET measurements, the AAS data suggests that high levels of Tl⁺ can perturb K⁺ transport and homeostasis. The absorption of Tl⁺ from the gut to hemolymph, measured by SIET, was confirmed by hemolymph measurements of Tl⁺ using AAS. This indicated that Tl⁺ gains access to the hemolymph and that sensitive tissues (such as the nervous system) are thus exposed. However, survival of <em>C. riparius</em> at these concentrations implies efficient mechanisms for detoxification of Tl⁺. This tolerance may involve sequestration in the gut, metal-binding proteins and increased secretion by the anal papillae and MTs. In addition, loss of K⁺ from the muscle may prevent hypokalemia in the hemolymph and gut.</p> / Master of Science (MSc)

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).