Anterior Malpighian Tubules Research Articles

An X-ray micro-computer tomography study of the Malpighian tubules of the Blue Bottle Blow Fly (Calliphora vomitoria) Diptera: Calliphoridae

Malpighian tubules are the insect equivalent of mammalian kidneys and normally drain into the gut at the junction between the mid and hind gut. The Malpighian tubules of the fruit fly Drosophila melanogaster are increasingly being used as a model for studying human renal tract development, histology, nephrolithiasis and urolithiasis. In the present study we report when using X-ray micro-computer tomography techniques, the larval, intrapuparial and adult stages of the larger Calliphora vomitoria can contain large amounts of calcium-rich concretions which are tightly packed in the lumen of both anterior Malpighian tubules. We show that it is feasible to utilise these calcium-rich concretions as a form of marking agent to delineate the various developmental stages of the Malpighian tubules including the crucial phase when the Malpighian tubules reconnect with the hind gut. In the majority of cases during the intrapuparial period the ureters of the Malpighian tubules did not start to re-canalise and thus reconnect with the developing hind gut until the 7th day of the 10-11 day. Just prior to ecdysis, virtually all the radio-opaque concretions in the Malpighian tubules had emptied into the hind gut and had then been completely excreted by the time the imago emerged from its puparium. In contrast, we show that in flies developing from larvae previously stained by ingesting Rhodamine B, a known substrate for both the Multi Xenobiotic Resistance and Multi Drug Resistant membrane transport systems, the efficiency with which these calcium-rich concretions are excreted by the imago as it emerges from its intrapuparial period can be significantly impaired. Therefore, it might be useful to include C. vomitoria as a model when studying renal tract development and urolithiasis using X-ray micro-computer tomography.

Major differences in the larval anatomy of the digestive and excretory systems of three Oestridae species revealed by micro-CT.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro-computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a 'glandular band' by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio-opaque concretions. Moreover, the anatomy of O. ovis third-instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a 'band' and anterior Malpighian tubules narrowly uniform throughout their entire length.

MP34-07 INTRAVITAL IMAGING OF THE DROSOPHILA MELANOGASTER MODEL OF HUMAN NEPHROLITHIASIS

You have accessJournal of UrologyStone Disease: Basic Research II1 Apr 2015MP34-07 INTRAVITAL IMAGING OF THE DROSOPHILA MELANOGASTER MODEL OF HUMAN NEPHROLITHIASIS Sohrab Naushad Ali, Dajung Kim, Thomas Tailly, Hassan Razvi, and Hon Leong Sohrab Naushad AliSohrab Naushad Ali More articles by this author , Dajung KimDajung Kim More articles by this author , Thomas TaillyThomas Tailly More articles by this author , Hassan RazviHassan Razvi More articles by this author , and Hon LeongHon Leong More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1303AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Drosophila Melanogaster (DM), a powerful translational model for many human diseases has recently emerged as a viable model of human nephrolithiasis. We have developed a novel bone and hydroxyapatite-specific fluorescent probe, Alendronate-FITC along with its negative control, Notdronate-FITC, for imaging calcium containing stones in vitro and in vivo. We investigated the utility of these probes in studying calcium oxalate stone formation in DM malpighian tubules (MT). METHODS Wild type DM were reared in a climate controlled room, temperature 22-25° C, humidity 40% and a 12 hour light/dark cycle. They were divided into groups and fed either a standard corn meal based diet or lithogenic diets containing 0.5% (w/v) Ethylene glycol or 0.5% (w/v) Sodium Oxalate. At 3 weeks DM were sampled from each group, the MT's dissected and incubated with Alendronate-FITC, Notdronate-FITC and PBS as a control. The MT's were then imaged using a confocal microscope. RESULTS DM fed the sodium oxalate diet showed more consistent stone formation compared to DM fed the ethylene glycol diet. DM fed the standard diet and stained with Alendronate-FITC showed scattered fluorescence in the anterior MT, most likely due to staining of its natural calcium concretions (Figure 1, bottom left panel). DM fed either of the lithogenic diets showed concentrated fluorescence throughout the MT (Figure 1, top left panel), indicating the presence of calcium oxalate stones when compared to in vitro stained hydroxyapatite nanoparticles stained with Alendronate-FITC. DM fed lithogenic diets and stained with Notdronate-FITC showed weak fluorescence with no signal in the lumen of MT's (Figure 1, top right panel). CONCLUSIONS Calcium oxalate stones can successfully be imaged using bisphosphonate based fluorescent probes. The superiority of these probes over conventional imaging techniques lies in the fact that they can be used for non-invasive in vivo imaging of stone formation. DM diets containing Alendronate-FITC may be used for imaging and quantifying the effects of different therapies on stone formation. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e412-e413 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sohrab Naushad Ali More articles by this author Dajung Kim More articles by this author Thomas Tailly More articles by this author Hassan Razvi More articles by this author Hon Leong More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

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 ∼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 ∼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.

Calcium homeostasis in larval and adult Drosophila melanogaster.

Calcium homeostasis in Drosophila melanogaster was examined in response to the challenges imposed by growth, reproduction and variations in dietary calcium content. Turnover time for calcium, calculated as the time for (45)Ca(2+)to accumulate to half the steady state value of 3.46 nmol/fly, was 3.3 days. Although larvae weighed 2x as much as adults, they contained 3-4x as much calcium. Anterior Malpighian tubules (Mts) contain much more calcium than posterior Mts, accounting for 25-30% of the calcium content of the whole fly. In response to a 6.2-fold increase in dietary calcium level, calcium content of whole flies increased only 10%. Hemolymph calcium concentration ( approximately 0.5 mM) was similar in males and females and in animals raised on diets differing in calcium content. Fluid secretion rate, secreted fluid calcium concentration, and transepithelial calcium flux in tubules isolated from flies raised on high and low calcium diets did not differ significantly. Malpighian tubules secrete calcium at rates sufficient to eliminate whole body calcium content in 0.5 and 3 days for tubules secreting fluid at basal and maximal rates, respectively. It is suggested that flies absorb high quantities of calcium from the diet and maintain homeostasis through the combined effects of elimination of calcium in fluid secreted by the Malpighian tubules and the sequestration of calcium in granules, especially within the distal segment of the anterior pair of Malpighian tubules.

Two types of concretions in Drosophila Malpighian tubules as revealed by X-ray microanalysis: A study on urine formation

In the Malpighian tubules of Drosophila two types of luminal concretions are formed. Type-I concretions mainly accumulate high quantities of calcium and magnesium in a matrix of proteoglycans; type-II concretions accumulate potassium. Higher quantities of organic matrix are found in type-II concretions than in type I concretions. The latter are stored in the distal segment of the anterior Malpighian tubules while type-II concretions are formed between apical microvilli in the transitional and the middle segment of anterior tubules and in the distal and the middle segment of posterior tubules. In the tubules of the pupa and in the first meconium to be produced, the magnesium and calcium contents of excreted type-I concretions are similar. The outer envelope of the puparium exhibits only traces of calcium in contrast to the cuticle of the larva. Both types of concretions are products of excretion: they eliminate calcium and magnesium or potassium, respectively. During winter time and under normal photoperiods both types of concretions can join together to form large “biocrystals” inhibiting the flow of urine. Concretion formation is discussed as an economical method to excrete high amounts of cations in a condensed form with the loss of little water.

Transport of the cationic fluorochrome rhodamine 123 in an insect's Malpighian tubule: indications of a reabsorptive function of the secondary cell type.

The pathway of rhodamine 123 was examined after injection into Sarcophaga flies and after in vitro labeling of the Malpighian tubules. After in vitro labeling the primary cells only retained this potential-sensitive dye for a short period while all secondary cells accumulated the dye from the tubule lumen. In vivo the secondary cells also accumulated rhodamine 123 from the lumen, but the primary cells in the distal parts of all four tubules retained the dye for prolonged periods. This was most pronounced in the distal part of the anterior Malpighian tubules, where rhodamine 123 was eventually precipitated on the luminal concretions. Rhodamine 123 initially accumulated in the secondary cell mitochondria and eventually in intensely fluorescing vesicles, probably lysosomes. No evidence for endocytotic processes from the lumen was found using Lucifer Yellow CH, fluorescent dextrans and fluorescent albumin. Prior incubation with the ionophores valinomycin, nigericin, CCCP (all 1 micrograms/ml), dinitrophenol (1 mM) and NaN3 (10(-2) M) inhibited the selective accumulation of rhodamine 123 to a large extent while monensin (1-5 micrograms/ml) showed little inhibitory effect. Furthermore, only cationic and no anionic or neutral dyes were accumulated by the secondary cells. In the fleshfly Calliphora and the fruitfly Drosophila, the dye rhodamine 123 also selectively accumulated in the secondary cells, as well in vitro as in vivo.

Changes in cytoskeletal actin patterns in the Malpighian tubules of the fleshfly, Sarcophaga bullata (Parker) (Diptera : Calliphoridae), during metamorphosis

Using the rhodamine-labelled phalloidin staining method in combination with detergent extraction, metamorphic changes in actin filament patterns were investigated in the Malpighian tubules of the fleshfly, Sarcophaga bullata (Parker) (Diptera : Calliphoridae). Metamorphosis in this organ implies a process of dedifferentiation, followed by a process of redifferentiation. During dedifferentiation, the large basal actin bundles of the primary cells disappear and the microvillar membrane surface of these cells decreases. Concomitantly, several vesicles are pinched off from infoldings of the brush border. In older pupae, the Malpighian tubules redifferentiate to give rise to adult tubules with actin patterns similar to those of larvae. During redifferentiation of the tubules, the secondary cells display a marked increase in the number of actin filaments in their protrusions. The primary cells in the distal part of the anterior Malpighian tubules of late pupae display a well-developed basal pattern of thick parallel actin bundles. In most cases, major changes in actin filament patterns are found simultaneously with major changes in cell shape, indicating a close relationship between these actin filaments and the process of cellular remodelling.

Morphology and biochemical composition of mineralized granules from the malpighian tubules of Musca autumnalis de geer larvae (Diptera: Muscidae)

Musca autumnalis De Geer (face fly) larvae form numerous small mineralized granules in the lumen of the anterior Malpighian tubules. The granules are spherical, ranging in diameter from 0.2 to > 10 μm and are composed of many concentric thin layers. The layers appear to be covered by an organic-rich material that may help to bind adjacent layers together. Individual layers appear to be heterogeneous with some composed of small rounded particles of inorganic-rich material imbedded in a loose fibrous mat while other layers appear more solid. The granules are primarily inorganic in composition (>85% ash) with calcium, phosphorus, and magnesium contributing more than 98% of the inorganic elements. Water content is approx. 10%. Carbonate carbon and hydrogen account for 2 to 3% of the granule weight. Nitrogen and organic carbon are only minor elements primarily present in the form of amino acids which contribute only 0.4% of the total granule weight. Aspartic acid and glutamic acid comprise more than 30% of the total amino acids detected after acid hydrolysis. The properties of face fly granules and puparial cuticle are compared, together with those of mineralized granules from other species of invertebrates.

Carbonic anhydrase in the face fly, Musca autumnalis (degeer) (Diptera: Muscidae)

Carbonic anhydrase was detected in the face fly, Musca autumnalis, and the development profile of the enzyme showed that maximum enzymatic activity occurred in the post-feeding larva. By comparison, little carbonic anhydrase activity was observed in post-feeding larvae of the house fly, Musca domestica. The enzyme was localized in the haemolymph, salivary glands, midgut and brain of post-feeding face fly larvae using an electrometric assay and Hansson's histochemical technique. No enzymatic activity was detected in the anterior Malpighian tubules, posterior Malpighian tubules, fat body, tracheae and carcass. These findings suggest that carbonic anhydrase has no function in the formation of the calcospherites located in the anterior Malpighian tubules of post-feeding larvae, but may be important in the ecdysial elimination of minerals during pupariation.

Calcium Storage in Face Fly (Diptera: Muscidae) Larvae for Puparium Formation1

The Malpighian tubules are shown to be the main storage site for Ca and other elements for puparium formation by the face fly, Musca autumnalis De Geer. Calcium in the anterior Malpighian tubules is stored in the form of amorphous, layered spheres 0.4 μm to >10.0 μm in diameter. Major elements in the composition of these spheres were Ca, P, and Mg, with traces of K. The weight of the tubules' content increased during the prepupal period. A major decrease then occurred and continued through the pupal period. The decrease results from movement of mineralized elements from the spheres and corresponds with the first significant amount of Ca and P found in the cuticle of the puparium. Mineralized spheres undergo dissolution in the proximal portion of the anterior Malpighian tubules, as indicated by external shrinkage and dissolution of internal layers, probably by changes in pH or enzymatic action.

PIGMENTED FAT CELLS IN A MUTANT OF DROSOPHILA MELANOGASTER

1. Cytoplasmic pigment granules are found in some of the fat cells of the recessive mutant, rc, of Drosophila melanogaster. These scattered red fat cells are located chiefly in the thorax and head, but a few occur in the abdomen and appendages of the adult. Using genetic methods it had been shown previously that these pigment accumulations are related to the synthesis of the brown eye pigment of this insect (Jones and Lewis, 1957).2. The pigmentation in the rc fat cells is suppressed when the rc gene is combined with the recessive factor, tuw. This combination, however, in no way alters the expression of the characteristic pattern of tuW as revealed by the presence of melanotic tumors in the caudal fat masses of the homozygous tuwrc flies. After a period of larval starvation, the tuwrc flies develop both the red-pigmented fat cells and melanotic tumors. The time of appearance of the rc pigment has been shifted under these nutritional and genetic conditions. The cytoplasmic pigment granules appear in the cells originating from the anteriormost section of the larval fatbody which is closely associated with the anterior Malpighian tubules. During the reorganization accompanying metamorphosis from the larval to the adult stage, these cells are redistributed mostly to the thoracic and cephalic regions while a few are found in the abdomen and appendages. An explanation is thus provided for the cytodifferentiation of pigmented and nonpigmented fat cells found side by side in the adult fly.3. The nature of the pigment granules has been examined in in vitro preparations at each of these periods of development, and of particular interest is the internal threadlike structure of these cytoplasmic inclusions during the early stages of pigment formation.