Calcium distribution during egg development in Calliphora vicina

Calcium distribution during egg development in Calliphora vicina has been measured by atomic absorption spectroscopy. When female flies are fed meat, their calcium content rises and then remains stable throughout egg development. As the ovaries accumulate calcium, there is a corresponding decrease in the calcium content of the Malpighian tubules but not in any other tissues. Tracer studies with ⁴⁵Ca indicate that calcium absorption and excretion continue throughout much of the egg cycle.

It is concluded that calcium absorption by female meat-fed flies exceeds the demands of the developing ovaries, and that appreciable amounts of calcium are excreted by the Malpighian tubules. In addition, the tubules may store calcium for later movement to the ovaries. Some of the practical problems associated with demonstrating such a storage role for the Malpighian tubules are discussed.     

Calcium transport by isolated anterior and posterior Malpighian tubules of Drosophila melanogaster: roles of sequestration and secretion

Ca(2+) transport was examined in isolated Malpighian tubules (MTs) of adult Drosophila melanogaster. All segments of both anterior and posterior MTs have substantial capacity to transport Ca(2+) and to play a role, therefore, in calcium homeostasis and elimination of excess dietary Ca(2+). Approximately 85% of Ca(2+) which enters the tubule is sequestered, and approximately 15% is secreted in soluble form into the tubule lumen. Tubules secreting fluid at maximal rates can remove an amount of Ca(2+) equal to the whole animal calcium content in approximately 9 h. Distal segments of the pair of anterior MTs can sequester the same amount of Ca(2+) in <2 h. Functional advantages of high Ca(2+) turnover rates are discussed. Transepithelial Ca(2+) secretion is increased by treatments which depolarize the transepithelial potential (thapsigargin, high K(+)), or acidify the secreted fluids (bicarbonate-free salines). The effects of pharmacological reagents and variations in bathing saline ionic composition indicate that the processes of secretion and sequestration are controlled independently, and that diltiazem-sensitive Ca(2+) channels are an important component of sequestration. The contribution of some form of apical Ca(2+) pump is evaluated.     

Storage-excretion of metallic cations in the adult housefly, Musca domestica

The objective of this investigation was to elucidate further the phenomenon of storage-excretion of metallic cations in the housefly. The sites for deposition of zinc, calcium and copper have been identified in this study. The metal intake of the flies was altered by raising one group on sucrose and tap water while in the experimental groups sucrose was supplemented with either 0.05% zinc sulphate, 0.05% calcium phosphate or 0.03% copper sulphate. There were no significant differences in the average life spans of the flies in different groups indicating physiological tolerance to the higher mineral intake. The Malpighian tubules, the midgut and the remainder of the body were analyzed for mineral content in houseflies ranging from 1 to 25 days posteclosion by atomic absorption spectrophotometry. There was a progressive age-associated increase in the total metal content of the flies with age. Zinc and calcium were primarily stored in the Malpighian tubules whereas copper was sequestered in the midgut. Microscopic examination of the epithelial cells of the Malpighian tubules and the midgut revealed a corresponding age-associated increase in mineralized concretions.     

Osmoregulation in a nectar-feeding insect, the carpenter bee Xylocopa capitata: water excess and ion conservation

Abstract The liquid diet and high metabolic water production during flight in the carpenter bee Xylocopa capitata Smith 1854 (Hymenoptera, Anthophoridae) causes a water excess, and this is exacerbated by a low dietary intake of ions. The nectar and pollen of the preferred food-plants, Virgilia divaricata Adamson and Podalyria calyptrata Willd., and other Fabaceae had low levels of sodium. Analyses of the bees and their body fluids showed that the bees have an exceptionally low Na content, and Na homeostasis seems to depend on recycling almost all Na which enters the rectum. The copious dilute urine (137 mOsm) had Na and K concentrations of only 3.4 and 7.0 ITIM, respectively. Isolated preparations of Xylocopa Malpighian tubules secreted a fluid with a K concentration 10 times that of the haemolymph. This means that recycling of K is as important as that of Na, and the bulk of K resorption probably occurs passively in the ileum. This study is the first to examine hymenopteran Malpighian tubules. Their stimulation by cAMP is indicative of the presence of a diuretic hormone in Xylocopa.     

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.     

Proteomic changes in response to crystal formation in Drosophila Malpighian tubules

Kidney stone disease is a major health burden with a complex and poorly understood pathophysiology. Drosophila Malpighian tubules have been shown to resemble human renal tubules in their physiological function. Herein, we have used Drosophila as a model to study the proteomic response to crystal formation induced by dietary manipulation in Malpighian tubules. Wild-type male flies were reared in parallel groups on standard medium supplemented with lithogenic agents: control, Sodium Oxalate (NaOx) and Ethylene Glycol (EG). Malpighian tubules were dissected after 2 weeks to visualize crystals with polarized light microscopy. The parallel group was dissected for protein extraction. A new method of Gel Assisted Sample Preparation (GASP) was used for protein extraction. Differentially abundant proteins (p<0.05) were identified by label-free quantitative proteomic analysis in flies fed with NaOx and EG diet compared with control. Their molecular functions were further screened for transmembrane ion transporter, calcium or zinc ion binder. Among these, 11 candidate proteins were shortlisted in NaOx diet and 16 proteins in EG diet. We concluded that GASP is a proteomic sample preparation method that can be applied to individual Drosophila Malpighian tubules. Our results may further increase the understanding of the pathophysiology of human kidney stone disease.     

Fluid secretion by in vitro preparations of the Malpighian tubules of the desert locust Schistocerca gregaria

In vitro preparations of locust Malpighian tubules can conveniently be made by a new technique in which the alimentary canal to which the tubules attach is removed from the insect and set up in Ringer's solution under liquid paraffin. Such Malpighian tubules will secrete a fluid iso-osmotic to the bathing fluid at a steady rate of about 1 to 2 nl min^?1 for some hours. The secreted fluid is rich in potassium ions, the lumen is at a potential positive to that of the bathing solution, and the rate of secretion can be controlled by changing the potassium concentration of the bathing fluid. It seems likely, therefore, that an active transport of potassium drives secretion ny locust Malpighian tubules. The secreted fluid contains an elevated concentration of phosphate ions. The Malpighian tubules will secrete at a high rate in a chloride-free phosphate-based solution. The rate of fluid secretion can be increased by treatment with cyclic AMP but 5-hydroxytryptamine has no such effect.     

Secretion of Na+, K+ and fluid by the Malpighian (renal) tubule of the larval cabbage looper Trichoplusia ni (Lepidoptera: Noctuidae)

The Malpighian (renal) tubules play important roles in ionic and osmotic homeostasis in insects. In Lepidoptera, the Malpighian tubules are structurally regionalized and the concentration of Na⁺ and K⁺ in the secreted fluid varies depending on the segment of tubule analyzed. In this work, we have characterized fluid and ion (Na⁺, K⁺, H⁺) transport by tubules of the larval stage of the cabbage looper Trichoplusia ni; we have also evaluated the effects of fluid secretion inhibitors and stimulants on fluid and ion transport. Ramsay assays showed that fluid was secreted by the iliac plexus but not by the yellow and white regions of the tubule. K⁺ and Na⁺ were secreted by the distal iliac plexus (DIP) and K⁺ was reabsorbed in downstream regions. The fluid secretion rate decreased > 50% after 25 μM bafilomycin A1, 500 μM amiloride or 50 μM bumetanide was added to the bath. The concentration of K⁺ in the secreted fluid did not change, whereas the concentration of Na⁺ in the secreted fluid decreased significantly when tubules were exposed to bafilomycin A1 or amiloride. Addition of 500 μM cAMP or 1 μM 5-HT to the bath stimulated fluid secretion and resulted in a decrease in K⁺ concentration in the secreted fluid. An increase in Na⁺ concentration in the secreted fluid was observed only in cAMP-stimulated tubules. Secreted fluid pH and the transepithelial electrical potential (TEP) did not change when tubules were stimulated. Taken together, our results show that the secretion of fluid is carried out by the upper regions (DIP) in T. ni Malpighian tubules. Upper regions of the tubules secrete K⁺, whereas lower regions reabsorb it. Stimulation of fluid secretion is correlated with a decrease in the K⁺/Na⁺ ratio.     

Effects of the microbial metabolite destruxin A on ion transport by the gut and renal epithelia of Drosophila melanogaster

Destruxins have been implicated in the infection process by entomopathogenic fungi and have been also found to be highly toxic when applied topically or ingested by different insect species. To gain insight into the mechanism of action of this toxin on insect internal organs, we have evaluated the effects of destruxin A on Drosophila melanogaster Malpighian tubules and gut tissues. Destruxin A was toxic when injected into adults; the calculated EC₅₀ was 0.11 mM. Destruxin A significantly inhibited fluid secretion rate by Malpighian tubules as well; the calculated IC₅₀ was 0.25 μM. The Na⁺ concentration in the secreted fluid increased significantly when tubules were exposed to 0.25 μM destruxin A, whereas pH and the concentrations of Ca²⁺ and K⁺ did not change. In gut, there was no effect of destruxin on H⁺ flux, but there was a significant decrease in K⁺ and Ca²⁺ absorption. The concentration of Ca²⁺ and K⁺ in the hemolymph of destruxin A‐injected flies was not significantly different from those of control flies after 3 h. Taken together, these results show that destruxin A produces differential effects on ion transport by renal and gut tissues. © 2012 Wiley Periodicals, Inc.     

Na(+)/H(+) exchange in mosquito Malpighian tubules

Fluid secretion and intracellular pH were measured in isolated mosquito Malpighian tubules to determine the presence of Na(+)/H(+) exchange. Rates of fluid secretion by individual Malpighian tubules in vitro were inhibited by 78% of control in the presence of 100 microM 5-(N-ethyl-n-isopropyl)-amiloride (EIPA), a specific inhibitor of Na(+)/H(+) exchange. Steady-state intracellular pH was measured microfluorometrically by using 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in individual Malpighian tubules. Bathing the Malpighian tubules in 0 mM extracellular Na(+) or in the presence of 100 microM EIPA reduced the steady-state intracellular pH by 0.5 pH units. Stimulation of the Na(+)/H(+) exchanger by using the NH(4)Cl pulse technique resulted in a rate of recovery from the NH(4)Cl-induced acute acid load of 8.7 +/- 1.0 x 10(-3) pH/s. The rates of recovery of intracellular pH after the acute acid load in the absence of extracellular Na(+) or in the presence of 100 microM EIPA were 0.7 +/- 0.6 and -0.3 +/- 0.3 x 10(-3) pH/s, respectively. These results indicate that mosquito Malpighian tubules possess a Na(+)/H(+) exchanger.     

The influence of canavanine and related compounds on the water balance system of locusts

In vivo increase in haemolymph volume of canavanine-treated locusts substantiates our previous in vitro findings that canavanine inhibits fluid secretion by locust Malpighian tubules. Furthermore when diuretic hormone is applied in vivo after canavanine treatment haemolymph volume is drastically reduced below levels retained in locusts untreated with canavanine. Again this is in accord with canavanine potentiation of semi-isolated Malpighian tubules and enhanced fluid secretion in vitro. The response is specific to canavanine; compounds similar in structure (arginine, argininic acid, citrulline, canaline, ornithine and homoserine) have no effect on the rate of fluid secreted by Malpighian tubules. Only partial competition is obtained with uridine homoserine.     

The action of the excretory apparatus of Calliphora vomitoria in handling injected sugar solution.

Recent evidence suggests that the isolated Malpighian tubules of Calliphora possess mechanisms which restrict the loss of glucose and trehalose from the insect. This report establishes that the intact, diuresing fly does not excrete glucose or trehalose when solutions of these sugars are injected. When solutions of non-metabolized sugars such as sorbose and xylose are injected into the fly, these sugars are rapidly excreted. High concentrations of sorbose and xylose are found in the urine, suggesting that rapid reabsorption of fluid occurs in the excretory apparatus even during the diuresis which the injections provoke. However, injected sucrose is apparently not excreted in large amounts and it is possible that the Malpighian tubules when functioning in vivo are impermeable to disaccharides.     

Developmental changes in Malpighian tubule cell structure.

Structural changes which occur in the Malpighian tubule yellow region primary cells during larval-pupal-adult development of the skipper butterfly Calpodes ethlius are described. The developmental changes in cell structure are correlated with functional changes in fluid transport (Ryerse, 1978a) in a way which supports osmotic gradient models of fluid secretion. Larval tubules are specialized for fluid secretion with deep basal infolds and elongate mitochondria-containing apical microvilli which provide channels in which osmotic gradients could be set up. The Malpighian tubule cells are extensively remodelled at pupation when fluid transport is switched off, but they persist intact through metamorphosis. At this time, the basement membrane doubles in thickness, the mitochondria are retracted from the microvilli and are isolated for degradation in autophagic vacuoles, and both apical and basal plasma membranes are internalized via coated vesicles for degradation in multivesicular bodies, which results in the shortening of the microville and the disappearance of the basal infolds. Mitochondria are re-inserted into the microvilli, and the basal infolds re-form in pharate adult stage Malpighian tubules when fluid secretion resumes. Adult tubules are similar in general structure to larval tubules and contain mitochondria in the microvilli and basal infolds. However, they differ from larval tubules in that they are capable of very rapid fluid transport, have a reduced tubule diameter and tubule wall thickness, a much thicker basement membrane and peripherally associated tracheoles. Mineral concretions of calcium phosphate accumulate in larval tubules, persist through metamorphosis and decline in number in adults, suggesting they serve some anabolic role.     

The corticotropin-releasing factor-like diuretic hormone 44 (DH44) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster

Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in insects, and are under neuroendocrine control by multiple neuropeptides secreted by identified neurons. Here, we demonstrate roles for CRF-like diuretic hormone 44 (DH₄₄) and Drosophila melanogaster kinin (Drome-kinin, DK) in desiccation and starvation tolerance.Gene expression and labelled DH₄₄ ligand binding data, as well as highly selective knockdowns and/or neuronal ablations of DH₄₄ in neurons of the pars intercerebralis and DH₄₄ receptor (DH₄₄-R2) in Malpighian tubule principal cells, indicate that suppression of DH₄₄ signalling improves desiccation tolerance of the intact fly.Drome-kinin receptor, encoded by the leucokinin receptor gene, LKR, is expressed in DH₄₄ neurons as well as in stellate cells of the Malpighian tubules. LKR knockdown in DH₄₄-expressing neurons reduces Malpighian tubule-specific LKR, suggesting interactions between DH₄₄ and LK signalling pathways.Finally, although a role for DK in desiccation tolerance was not defined, we demonstrate a novel role for Malpighian tubule cell-specific LKR in starvation tolerance. Starvation increases gene expression of epithelial LKR. Also, Malpighian tubule stellate cell-specific knockdown of LKR significantly reduced starvation tolerance, demonstrating a role for neuropeptide signalling during starvation stress.     

Organically Grown Food Provides Health Benefits to Drosophila melanogaster

The “organic food” market is the fastest growing food sector, yet it is unclear whether organically raised food is nutritionally superior to conventionally grown food and whether consuming organic food bestows health benefits. In order to evaluate potential health benefits of organic foods, we used the well-characterized fruit fly Drosophila melanogaster as a model system. Fruit flies were raised on a diets consisting of extracts of either conventionally or organically raised produce (bananas, potatoes, raisins, soy beans). Flies were then subjected to a variety of tests designed to assess overall fly health. Flies raised on diets made from organically grown produce had greater fertility and longevity. On certain food sources, greater activity and greater stress resistance was additionally observed, suggesting that organic food bestows positive effects on fly health. Our data show that Drosophila can be used as a convenient model system to experimentally test potential health effects of dietary components. Using this system, we provide evidence that organically raised food may provide animals with tangible benefits to overall health.     

Segment-specific Ca2+ transport by isolated Malpighian tubules of Drosophila melanogaster: A comparison of larval and adult stages

Haemolymph calcium homeostasis in insects is achieved through the regulation of calcium excretion by Malpighian tubules in two ways: (1) sequestration of calcium within biomineralized granules and (2) secretion of calcium in soluble form within the primary urine. Using the scanning ion-selective electrode technique (SIET), basolateral Ca²⁺ transport was measured at the distal, transitional, main and proximal tubular segments of anterior tubules isolated from both 3rd instar larvae and adults of the fruit fly Drosophila melanogaster. Basolateral Ca²⁺ transport exceeded transepithelial secretion by 800-fold and 11-fold in anterior tubules of larvae and adults, respectively. The magnitude of Ca²⁺ fluxes across the distal tubule of larvae and adults were larger than fluxes across the downstream segments by 10 and 40 times, respectively, indicating a dominant role for the distal segment in whole animal Ca²⁺ regulation. Basolateral Ca²⁺ transport across distal tubules of Drosophila varied throughout the life cycle; Ca²⁺ was released by distal tubules of larvae, taken up by distal tubules of young adults and was released once again by tubules of adults ⩾168 h post-eclosion. In adults and larvae, SIET measurements revealed sites of both Ca²⁺ uptake and Ca²⁺ release across the basolateral surface of the distal segment of the same tubule, indicating that Ca²⁺ transport is bidirectional. Ca²⁺ uptake across the distal segment of tubules of young adults and Ca²⁺ release across the distal segment of tubules of older adults was also suggestive of reversible Ca²⁺ storage. Our results suggest that the distal tubules of D. melanogaster are dynamic calcium stores which allow efficient haemolymph calcium regulation through active Ca²⁺ sequestration during periods of high dietary calcium intake and passive Ca²⁺ release during periods of calcium deficiency.     

Diuresis in a desert beetle? hormonal control of the malpighian tubules ofOnymacris plana (Coleoptera: Tenebrionidae)

Summary Malpighian tubules of a desert tenebrionid beetle,Onymacris plana, have been studied as isolated preparations. Under control conditions tubules of female beetles secreted fluid at an average rate of 3.3 nl/min, but this rate was increased 20–25 times by a diuretic hormone (DH).Homogenates of the brain, corpora cardiaca (CC) and prothoracic ganglion induced striking increases in tubule secretion rates, which sometimes exceeded 100 nl/min. The increased rates were sustained for 3 h without renewal of the medium. Diuretic activity was also present in the other thoracic ganglia. High K treatment caused release of DH from the CC only.Exogenous cyclic AMP (1 mM) stimulated the isolated tubules ofO. plana, but to a lesser extent than the DH. The cationic composition of the secreted fluid resembled that of most other insect tubules, with high K and low Na concentrations. Stimulation with DH doubled the Na concentration.The DH was not inactivated by the tubules themselves, but was destroyed by contact with the haemolymph. An inactivation mechanism is vital in the apparently contradictory situation of a desert beetle possessing a diuretic hormone. The role of the cryptonephric system during diuresis is unknown.Abbreviations DH diuretic hormone - cAMP adenosine 3:5-cyclic monophosphoric acid - CC corpora cardiaca     

A C-terminal aldehyde analog of the insect kinins inhibits diuresis in the housefly

The insect kinins are present in a wide variety of insects and function as potent diuretic peptides in flies. A C-terminal aldehyde insect kinin analog, Fmoc-RFFPWG-H (R-LK-CHO), demonstrates stimulation of Malpighian tubule fluid secretion in crickets, but shows inhibition of both in vitro and in vivo diuresis in the housefly. R-LK-CHO reduced the total amount of urine voided over 3 h from flies injected with 1 microL of distilled water by almost 50%. The analog not only inhibits stimulation of housefly fluid secretion by the native kinin Musdo-K, but also by thapsigargin, a SERCA inhibitor, and by ionomycin, a calcium ionophore. The activity of R-LK-CHO is selective, however, as related C-terminal aldehyde analogs do not demonstrate an inhibitory response on housefly fluid secretion. The selective inhibitory activity of R-LK-CHO on housefly tubules represents an important lead in the development of environmentally friendly insect management agents based on the insect kinins.     

Mechanisms of calcium sequestration by isolated Malpighian tubules of the house cricket Acheta domesticus

Hemolymph calcium homeostasis in insects is achieved by the Malpighian tubules, primarily by sequestering excess Ca²⁺ within internal calcium stores (Ca‐rich granules) most often located within type I (principal) tubule cells. Using both the scanning ion‐selective electrode technique and the Ramsay secretion assay this study provides the first measurements of basolateral and transepithelial Ca²⁺ fluxes across the Malpighian tubules of an Orthopteran insect, the house cricket Acheta domesticus. Ca²⁺ transport was specific to midtubule segments, where 97% of the Ca²⁺ entering the tubule is sequestered within intracellular calcium stores and the remaining 3% is secreted into the lumen. Antagonists of voltage‐gated (L‐type) calcium channels decreased Ca²⁺ influx ≥fivefold in adenosine 3′,5′‐cyclic monophosphate (cAMP)‐stimulated tubules, suggesting basolateral Ca²⁺ influx is facilitated by voltage‐gated Ca²⁺ channels. Increasing fluid secretion through manipulation of intracellular levels of cAMP or Ca²⁺ had opposite effects on tubule Ca²⁺ transport. The adenylyl cyclase‐cAMP‐PKA pathway promotes Ca²⁺ sequestration whereas both 5‐hydroxytryptamine and thapsigargin inhibited sequestration. Our results suggest that the midtubules of Acheta domesticus are dynamic calcium stores, which maintain hemolymph calcium concentration by manipulating rates of Ca²⁺ sequestration through stimulatory (cAMP) and inhibitory (Ca²⁺) regulatory pathways.