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The Keap1-Nrf2 pathway is a major upstream regulator of xenobiotic detoxification. In Drosophila, directed activation of the protein complex of Keap1 and CncC (the homolog of human Nrf2) in principal and stellate cells of the Malpighian (renal) tubules confers resistance to lethal doses of the pesticide malathion, which is metabolized into organic anions. Dietary exposure to organic anions such as salicylate (10 mM) causes increases in fluid secretion rate and salicylate flux across Malpighian (renal) tubules. Here we used salicylate-selective microelectrodes and Ramsay assays to determine the role of Keap1/CncC in regulating these responses. Fluid secretion rate and salicylate flux across tubules isolated from adults with directed activation of Keap1/CncC in the principal cells are comparable to the values from salicylate-fed controls. Fluid secretion rate, concentration of salicylate in the secreted fluid and salicylate flux did not differ significantly between tubules isolated from adults with directed activation of Keap1/CncC in the principal cells reared on a diet containing salicylate and those reared on control media, indicating that the detoxification pathway was activated regardless of the presence of dietary salicylate. This is in contrast to the significant increase in fluid secretion rate and salicylate flux between tubules isolated from salicylate-fed adults and adults reared on a control diet with directed activation of Keap1/CncC in the stellate cells, supporting previous studies that demonstrated the inability of stellate cells to transport organic anions. Taken together, these results suggest a role for Keap1/CncC in upregulating fluid secretion in response to the presence of dietary organic anions. / Thesis / Master of Science (MSc) / The Keap1-Nrf2 pathway is a major upstream regulator of xenobiotic detoxification. In Drosophila, directed activation of the protein complex of Keap1 and CncC (the Nrf2 homolog) in principal and stellate cells of the Malpighian (renal) tubules confers resistance to lethal doses of the pesticide malathion, which is metabolized into organic anions. Dietary exposure to organic anions such as salicylate (10 mM) causes increases in fluid secretion rate and salicylate flux across Malpighian (renal) tubules that are comparable to tubules isolated from adults with activated Keap1/CncC reared on a salicylate-free diet. This suggests a role for Keap1/CncC in upregulating fluid secretion in response to the presence of dietary organic anions.

Wang Jian Bin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 117-131). / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.x / Chapter PART I. --- GENERAL INTRODUCTION --- p.4 / Chapter PART II. --- LITERATURE REVIEW --- p.6 / Chapter A. --- The structure and functions of insect Malpighian tubules --- p.6 / Chapter I. --- The excretory system of insects --- p.6 / Chapter 1. --- Morphology of Malpighian tubules --- p.6 / Chapter 2. --- Common types of Malpighian tubule system --- p.7 / Chapter 3. --- Morphology of hindgut --- p.8 / Chapter II. --- Structure of insect Malpighian tubules --- p.9 / Chapter 1. --- General organization of the Malpighian tubules --- p.9 / Chapter 2. --- Structure of the principal cell --- p.10 / Chapter 3. --- The structure of other cell types --- p.14 / Chapter 4. --- The cryptonephridial systems in larvae of Lepidoptera and Coleoptera --- p.16 / Chapter III. --- Functions of insect Malpighian tubules --- p.18 / Chapter 1. --- Mechanism of fluid secretion by Malpighian tubules --- p.18 / Chapter 1.1. --- Ion transport --- p.18 / Chapter 1.2. --- Fluid transport --- p.19 / Chapter 2. --- Active transport of organic compounds by Malpighian tubules --- p.19 / Chapter 2.1. --- Organic anions --- p.19 / Chapter 2.2. --- Organic cations --- p.20 / Chapter 3. --- Resporptive processes in Malpighian tubules --- p.20 / Chapter 3.1. --- KC1 resorption --- p.20 / Chapter 3.2. --- Reabsorption of sugars --- p.21 / Chapter 4. --- The passive permeability of Malpighian tubules --- p.21 / Chapter B. --- The biology and mode of action of Bacillus thuringiensis --- p.23 / Chapter I. --- Introduction --- p.23 / Chapter II. --- Background --- p.23 / Chapter III. --- "Cytology of germination, outgrowth and sporulation" --- p.24 / Chapter IV. --- Bacillus thuringiensis and its toxins --- p.26 / Chapter V. --- Histopathological effects of Bacillus thuringiensis δ-endotoxin on Lepidopterous larva --- p.29 / Chapter VI. --- Mode of action of Bacillus thuringiensis δ-endotoxin --- p.32 / Chapter C. --- The biology and pathological effects of microsporidian protozoa --- p.36 / Chapter I. --- Life cycle of microsporidian protozoa --- p.36 / Chapter II. --- Germination of microsporidian protozoa --- p.37 / Chapter III. --- The fine structure of microsporidian protozoa --- p.38 / Chapter IV. --- Mass production and storage --- p.42 / Chapter V. --- Pathology of microsporidian protozoa --- p.44 / Chapter PART III. --- LIGHT AND ELECTRON MICROSCOPIC STUDIES OF THE MALPIGHIAN TUBULES OF PIERIS CANIDIA LARVA (LEPIDOPTERA) --- p.48 / Summary --- p.48 / Introduction --- p.48 / Materials and methods --- p.49 / Results --- p.50 / Discussion --- p.53 / Chapter PART IV. --- HISTOCHEMICAL STUDIES ON THE PIERIS CANIDIA LARVAL MALPIGHIAN TUBULES --- p.58 / Summary --- p.58 / Introduction --- p.59 / Materials and methods --- p.60 / Results --- p.52 / Discussion --- p.66 / Chapter PART V. --- SEPARATION AND PURIFICATION OF PARASPORAL CRYSTALS OF BACILLUS THURINGIENSIS VAR. KURSTAKI --- p.70 / Summary --- p.70 / Introduction --- p.70 / Materials and methods --- p.74 / Results --- p.77 / Discussion --- p.77 / Chapter PART VI. --- HISTOPATHOLOGICAL EFFECTS OF BACILLUS THURINGIENSIS VAR. KURSTAKI δ-ENDOTOXIN ON THE MALPIGHIAN TUBULES OF PIERIS CANIDIA LARVA --- p.79 / Summary --- p.79 / Introduction --- p.79 / Materials and methods --- p.81 / Results --- p.83 / Discussion --- p.86 / Chapter PART VII. --- THE FINE STRUCTURE OF A MICROSPORIDIAN NOSEMA POLYVORA FROM CABBAGE WHITE PIERIS CANIDIA --- p.92 / Summary --- p.92 / Introduction --- p.92 / Materials and methods --- p.94 / Results --- p.94 / Discussion --- p.97 / Chapter PART VIII. --- HISTOPATHOLOGICAL EFFECTS OF NOSEMA POLYVORA ON THE MALPIGHIAN TUBULES OF PIERIS CANIDIA LARVA --- p.103 / Summary --- p.103 / Introduction --- p.103 / Materials and methods --- p.105 / Results --- p.105 / Discussion --- p.107 / Chapter PART IX. --- GENERAL DISCUSSION --- p.111 / Chapter PART X. --- CONCLUSION AND SUMMARY --- p.115 / REFERENCES --- p.117 / FIGURES AND TABLES --- p.132

Thesis (Ph.D.)--McMaster University, 2006. / Supervisor: Michael J. O'Donnell. Includes bibliographical references (leaves 279-310).

Stimulation of urine production by the Malpighian (renal) tubules in Rhodnius prolixus is regulated by at least two diuretic hormones, CRF-related peptide and serotonin, that have traditionally been believed to function through the activation of cAMP-mediated intracellular second messenger pathways. In this study I demonstrate that serotonin stimulation triggered, in addition to cAMP, intracellular Ca2+ waves in the Malpighian tubule cells of R. prolixus. Treatment with the intracellular Ca2+ chelator BAPTA-AM blocked the intracellular Ca2+ waves and reduced serotonin-stimulated fluid secretion by 75%. This suggests a role for intracellular Ca2+ signaling in the excretory system of R. prolixus. Serotonin stimulated Malpighian tubules (MTs) exposed to Ca2+-free saline plus BAPTA-AM secreted an abnormal fluid, showing: increased K+ concentration, reduced Na+ concentration and lower pH. These results along with measurement of transepithelial potential (TEP) suggest that the basolateral Na+:K+:2Cl- cotransporter (NKCC) activity is reduced in tubule cells treated with BAPTA-AM, suggesting that Ca2+ is required to modulate the activity of the basolateral NKCC. Treatment with the non-hydrolysable cell-permeable cAMP analog, 8Br-cAMP, produced fluid with the same K+ and Na+ concentration and at the same secretion rate as serotonin-stimulated tubules. In addition, 8Br-cAMP triggered intracellular Ca2+ oscillations similar to those obtained with serotonin. 8Br-cAMP-stimulated tubules treated with BAPTA-AM decreased their fluid secretion by about 40% and increased Na+ concentration, similar to the effect observed on serotonin-stimulated tubules. Therefore, I conclude that the intracellular Ca2+ waves triggered by serotonin are mediated by cAMP. The role of inositol-3-phospate (InsP3) in Ca2+ release was tested by treating the tubules with the InsP3 receptor blocker xestospongin. The treatment decreased fluid secretion rate as well as the amplitude of Ca2+ waves in serotonin-stimulated tubules. These results suggest that serotonin activates the production of InsP3 and, most likely, diacylglycerol (DAG). Thus, I decided to test whether the protein kinase C (PKC) may be involved in serotonin-stimulated secretion. The PKC inhibitors chelerythrine and bisindolylmaleimide (BIM) decreased secretion fluid rate in serotonin-stimulated tubules by 50% and 70%, respectively. Fluid secreted by tubules treated with BIM showed no differences in K+ and Na+ concentrations compared to controls, however both ion fluxes decreased. The evidence suggests that PKC is involved in serotonin stimulated secretion; the mechanism is still not understood. Taken together, the results suggest that cAMP, Ca2+ and PLC-PKC pathway are involved in serotonin stimulated secretion. However cAMP stimulation is enough for maximal secretion rate. Therefore PLC-PKC must act downstream of cAMP. Based on those results we hypothesize that serotonin binds a GPCR, increasing cAMP by activation of an adenylate cyclase (AC). Subsequently, cAMP is somehow able to activate PLC, which finally produces Ca2+ release, PKC activation and NKCC upregulation.

<p>The transport of ammonia by various tissues throughout the body is of fundamental importance for nitrogen excretion in invertebrates, yet sites and mechanisms of ammonia transport are not presently well understood. In this thesis a novel ammonium-selective microelectrode was developed using the ionophore TD19C6, which is approximately 3800-fold more selective for NH₄⁺ than Na⁺ compared with the 100-fold difference of nonactin used in previous microelectrodes. We investigated the accuracy of the ammonium microelectrode in solutions simulating <em>Drosophila</em> haemolymph (25 mM K⁺) and secreted fluid (120 mM K⁺). In haemolymph-like solutions, ammonium could be measured down to about 1 mM, with an error of 0.5 mM, while in secreted fluid-like conditions ammonium could be determined to within 0.3 mM down to a level of 1 mM NH₄⁺ in the presence of 100 to 140 mM K⁺. These results suggested that the ammonium microelectrode could be used to measure ammonium in the presence of physiological levels of potassium, unlike previous studies. We also quantified ammonium secretion by the Malpighian (renal) tubules of larvae. Ammonium concentrations of secreted fluid were consistently equivalent to or above ammonium concentrations of bathing salines. With a lumen-positive transepithelial potential, these results suggested an active secretory mechanism for ammonia transport. Under conditions of low K⁺ concentrations, the ability of the tubules to concentrate ammonium in secreted fluid was significantly enhanced, indicating some level of competition between NH₄⁺ and K⁺ for common transporters. The new ammonium-selective microelectrode is sufficiently sensitive to detect ammonium at the picomol level.</p> / Master of Science (MSc)

Call number: LD2668 .T4 1985 E46 / Master of Science

The evolution of the blood feeding adaptation has required precise coordination of multiple physiological processes in the insect, such as reproduction, behavior, digestion and diuresis. These processes are under careful synchronous hormonal control. For rapid excretion, multiple diuretic hormones are known. Although originally described based on their ability to stimulate hindgut contractions, the Aedes kinins have been shown to stimulate fluid secretion in female mosquitoes of Aedes aegypti. Aedes kinins are leucokinin-like neuropeptides released from neurosecretory cells in the brain and abdominal ganglia. They act by binding to the Aedes kinin receptor, a G proteincoupled receptor (GPCR). The Aedes kinin receptor has been cloned, sequenced, functionally characterized, and immunolocalized to stellate cells in the Malpighian tubules of Ae. aegypti. In addition to their myotropic and diuretic roles, leucokinin-like peptides and/or their receptors have been also been discovered in the nervous, digestive, and reproductive systems of other arthropod species. Therefore, the Aedes kinins have the potential to function in several simultaneous physiological processes that are stimulated by blood feeding. This thesis aims to understand better their role in the whole mosquito by investigating the Aedes kinin receptor's global expression as well as its in vivo contribution to post-prandial diuresis. Presence of the Aedes kinin receptor was investigated in the head, posterior midgut (stomach), hindgut, ovaries, and Malpighian tubules of both non blood-fed and blood-fed females by western blot using anti-receptor antibodies. The receptor was then immunolocalized in the posterior midgut and rectum. Finally, RNAi was employed to knock down kinin receptor expression, followed by measurement of in vivo urine excretion post blood feeding in a precision humidity chamber. Transcript and protein knockdown were confirmed by qPCR and immunohistochemistry, respectively. Results indicate widespread expression of the Aedes kinin receptor protein in organs novel for hematophagous insects and demonstrate the receptor's fundamental role in rapid diuresis. These findings strongly point to the Aedes kinins as integrative signaling molecules that could coordinate multiple physiological systems. The Aedes kinins could therefore have contributed to the success of the blood feeding adapation in mosquitoes.