Non-apoptotic function of apoptotic proteins in the development of Malpighian tubules of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Drosophila metamorphosis is characterized by the histolysis of larval structures by programmed cell death, which paves the way for the establishment of adult-specific structures under the influence of the steroid hormone ecdysone. Malpighian tubules function as an excretory system and are one of the larval structures that are not destroyed during metamorphosis and are carried over to adulthood. The pupal Malpighian tubules evade destruction in spite of expressing apoptotic proteins, Reaper, Hid, Grim, Dronc and Drice. Here we show that in the Malpighian tubules expression of apoptotic proteins commences right from embryonic development and continues throughout the larval stages. Overexpression of these proteins in the Malpighian tubules causes larval lethality resulting in malformed tubules. The number and regular organization of principal and stellate cells of Malpighian tubules is disturbed, in turn disrupting the physiological functioning of the tubules as well. Strikingly, the localization of beta-tubulin, F-actin and Disclarge (Dlg) is also disrupted. These results suggest that the apoptotic proteins could be having non-apoptotic function in the development of Malpighian tubules.     

An ultrastructural study of Dirofilaria immitis infection in the Malpighian tubules of Anopheles quadrimaculatus

An ultrastructural study was conducted of the Malpighian tubules of Anopheles quadrimaculatus, both uninfected and following infection with Dirofilaria immitis. The Malpighian tubules in Anopheles are composed of primary and stellate cells. The primary cells are the predominant cell type and are characterized by the presence of membrane-bound, intracellular, mineralized concretions and large apical microvilli containing mitochondria. Following the infective blood meal, the microfilariae enter the primary cells of the Malpighian tubules and reside in the cytoplasm in a clear zone without a delimiting membrane. Cells in infected tubules differ from those in uninfected tubules in that the membranes of the vacuoles surrounding the concretions are disrupted in many specimens. The apical and basal cell membranes and the mitochondria associated with these are not disrupted during the first 6-8 days of infection. These observations differ sharply from those previously described in Aedes taeniorhynchus infected with D. immitis. The observations are consistent with the hypothesis that the extended transport capacity observed in previous physiological studies of An. quadrimaculatus infected with D. immitis are dependent on the prolonged normal ultrastructure of the apical microvilli, mitochondria, and basal membranes.     

Micromorphology of the malpighian tubules in the louse Pediculus humanus corporis (Anoplura)

The ultrastructure of the Malpighian tubes in human louse Pediculus humanus corporis has been studied. The cells of the Malpighian tubules have the uniform structure: the apical surface is covered with microvilli, the basal plasmatic membrana forms relatively small invaginations. The microvilli are most developed in cells of the proximal department of the Malpighian tubules. Microvilli of the apical surface of the cells do not contain mitochondria which are localized mainly in supranuclear part of the cell. Cells are lined with a homogenous basal membrane.     

Evidence and bioassay for diuretic factors in the nervous system of larvalHeliothis virescens

Summary Diuretic factors were studied in the central nervous system of larvae of the tobacco budworm,Heliothis virescens, using [¹⁴C]urea as a sensitive indicator for water movement through isolated Malpighian tubules. The assay required Na⁺ and a pH of 6.0–6.2 for maximum activity. Malpighian tubules had high secretory activity in feeding larvae of the fifth instar, but the activity declined during the burrowing-digging stage that preceded pupation. Malpighian tubules from starved larvae showed a greater response to extracts of nervous tissues than did tubules from feeding larvae, and extracts showed a dose-response relationship with fluid secretion. Diuretic activity was distributed throughout all parts of the central nervous system with the brain having the most activity. Brain extracts increased fluid secretion by in vitro Malpighian tubules by more than 3-fold and doubled the rate of dye clearance from the hemolymph in vivo. Diuretic activity in nervous tissue extracts was unaffected by boiling but sensitive to proteases. Fluid secretion by in vitro tubules was increased by cAMP, dbcAMP, theophylline, octopamine and dopa. These studies provide evidence for the presence of diuretic factors in the central nervous system ofH. virescens larvae and describe a sensitive bioassay for these factors.Abbreviations AR activation ratio - cAMP cyclic AMP - dbcAMP dibutyryl cyclic AMP - dbcGMP dibutyryl cyclic GMP - Dopa dihydroxyphenylalanine - 5-HT 5-hydroxytryptamine - L1 larval instar - VCNS ventral central nervous system     

Vasopressin-like peptide and its receptor function in an indirect diuretic signaling pathway in the red flour beetle

The insect arginine vasopressin-like (AVPL) peptide is of special interest because of its potential function in the regulation of diuresis. Genome sequences of the red flour beetle Tribolium castaneum yielded the genes encoding AVPL and AVPL receptor, whereas the homologous sequences are absent in the genomes of the fruitfly, malaria mosquito, silkworm, and honeybee, although a recent genome sequence of the jewel wasp revealed an AVPL sequence. The Tribolium receptor for the AVPL, the first such receptor identified in any insect, was expressed in a reporter system, and showed a strong response (EC(50)=1.5 nM) to AVPL F1, the monomeric form having an intramolecular disulfide bond. In addition to identifying the AVPL receptor, we have demonstrated that it has in vivo diuretic activity, but that it has no direct effect on Malpighian tubules. However, when the central nervous system plus corpora cardiaca and corpora allata are incubated along with the peptide and Malpighian tubules, the latter are stimulated by the AVPL peptide, suggesting it acts indirectly. Summing up all the results from this study, we conclude that AVPL functions as a monomer in Tribolium, indirectly stimulating the Malpighian tubules through the central nervous system including the endocrine organs corpora cardiaca and corpora allata. RNA interference in the late larval stages successfully suppressed mRNA levels of avpl and avpl receptor, but with no mortality or abnormal phenotype, implying that the AVPL signaling pathway may have been near-dispensable in the early lineage of holometabolous insects.     

Cytoskeletal F-actin patterns in whole-mounted insect Malpighian tubules.

The distribution of actin filaments in Malpighian tubules of the fleshfly Sarcophaga bullata (Parker) was investigated before and after metamorphosis by means of the rhodamine phalloidin staining method. The numerous primary cells show a pattern of thick basal actin bundles resembling stress fibres of cultured cells, while the apical microvillar zone shows a bright and homogeneous labelling. The less abundant stellate cells contain no such basal actin bundles and their apical microvillar zone gets only faintly stained. Late larval stages display fingerlike infoldings and an increased actin filament concentration at the apical membrane of the stellate cells. During metamorphosis the Malpighian tubules dedifferentiate and eventually redifferentiate to give rise to adult tubules resembling larval ones. The different types of actin filament organisation in the primary and stellate cells of the Malpighian tubules are discussed.     

Regulatory interactions and role in cell type specification of the Malpighian tubules by the cut, Krüppel, and caudal genes of Drosophila.

Krüppel and caudal genes are both required for normal segmentation of the embryo, and the developmental regulatory gene cut is necessary for the normal specification of external sensory organs. These three genes are also expressed in the Malpighian tubules before and during differentiation. Two of the genes, Krüppel and cut, are known to be required for development of the tubules. We report that the absence of maternal and zygotic caudal function reduces their normal growth and elongation. Normal Krüppel function, which is known to be required for caudal expression, is also required for cut expression, while cut and caudal are expressed independently of each other. Cell type transformations of Malpighian tubules were studied by examining the effects of mutations on the expression of markers specific to Malpighian tubules, hindgut, or midgut of normal embryos. Loss of Krüppel activity confers hindgut characteristics on those cells that normally form the Malpighian tubules with all markers tested. Loss of cut function alters the expression of some markers but not others. The pathway of tissue specific gene regulation, apparently, branches beyond Krüppel to form at least a cut and a caudal branch.     

Actin redistribution in mosquito malpighian tubules after a blood meal and cyclic AMP stimulation

Fluid secretion by mosquito Malpighian tubules is critical to maintaining fluid and electrolyte balance after a blood meal. Endogenous cAMP levels increase in Malpighian tubules after a blood meal. Here, we determined if corresponding changes in intracellular actin distribution occur after a blood meal or dibutyryl-cAMP (db-cAMP) stimulation and whether altering actin turnover inhibits secretion. In untreated Malpighian tubules, beta-actin immunostaining was more intense in the apical region of adult Malpighian tubules than in the cytoplasm. Stimulation by a blood meal or db-cAMP significantly decreased beta-actin immunostaining in the non-apical region of the cell. Db-cAMP had similar effects in larvae and pupae Malpighian tubules. In contrast, no detectable shift in F-actin distribution was detected; however, F-actin bundles within the cytoplasm increased in size after treatment with db-cAMP. Pretreatment of Malpighian tubules with agents perturbing actin fiber assembly and disassembly decreased basal secretion rates and inhibited the stimulatory effects of db-cAMP. Our results show (1) beta-actin redistributes toward the apical membrane after a blood meal and this correlates temporally with increase urine flow rate and intracellular cAMP levels, (2) Malpighian tubules from all developmental stages exhibit this same response to db-cAMP-stimulation, and (3) dynamic assembly and disassembly of beta-actin is required for db-cAMP-stimulated secretion.     

Electron-microscopic study of the excretory system of hungry females of the tick Hyalomma asiaticum P. Sch. et E. Schl. 2]

In H. asiaticum the cells of the Malpighian tubules and these of the rectal cas have the uniform structure: the apical surface is covered with microvilli, the basal plasmatic membrane forms relatively small invaginations. As to ultrastructural characters, there is no distinct division of the Malpighian tubule into departments. The distal ends of the tubules are not only somewhat enlarged and form the so-called ampulla cells of which are noticeably flattened. The microvilli and basal folds of the plasmatic membrane in this area of the tubule are indistinct. The cells of the ampulla and the neighbouring area of the tubule are characterized by the presence of inclusions with mucopolysaccharide secretion confined by the membrane. The microvilli are most developed on cells of the proximal ends of the Malpighian tubules. Well developed microvilli of the rectal sac form a striated border each containing a microtube inside. The basal invaginations are developed here better than in the cells of the Malpighian tubules.     

The fine structure of the light organ of the New Zealand glow-worm Arachnocampa luminosa (Diptera: Mycetophilidae).

The swollen distal tips of the Malpighian tubules of the glow-worm Arachnocampa luminosa constitute the light organ. The ventral and lateral surfaces are covered by a tracheal ‘reflector’ and the nervous supply to the light organ comes from the ganglion in the penultimate segment. Fine nerve terminals, axons, and glial cells can be seen in close proximity to the basal surface of the cells of the light organ. The epithelial cells of the light organ are large, the cytoplasm dense, homogeneous and acidophilic. The cytoplasm gives a strong positive reaction for protein. The cytoplasm contains a high density of free ribosomes, patches of dense material, smooth endoplasmic reticulum, glycogen and scattered microtubules. Mitochondria are numerous; they are large, randomly distributed and packed with fine cristae. These cells lack the features characteristic of Malpighian tubule epithelial cells; infolding of the apical and basal cell surfaces is reduced and the cytoplasm contains few organelles. These cells do not contain secretory or photocyte granules and the grainy cell matrix is thought to be the luciferin substrate. Oxygen is supplied via the tracheal layer (which may have secondary reflecting properties) and light production controlled by neurosecretory excitation either directly via synapses, or by hormones. There are no other reports of Malpighian tubules of insects producing light and the fine structure of these cells is distinct. Thus, the swollen distal tips of the Malpighian tubules of the glow-worm undoubtedly constitute a unique luminescent organ.     

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.     

Genetic control of development of the Malpighian vessels in Drosophila melanogaster

Malpighian tubules of insects are a functional analog of mammalian kidneys and serve as a classical model for studying the structure and functions of transport epithelium. The review contains the data on structural organization, functioning, and formation of the Malpighian tubules during embryogenesis in Drosophila melanogaster. Various systems of genes are described that control the program of development of the renal (Malpighian) tubules in D. melanogaster. A special attention is paid to the ways of signal transduction and factors involved in cell differentiation, proliferation, and morphological transformation during development of the Malpighian tubules. Evolutionarily conservative genetic systems are considered that are involved in the control of development of both the renal epithelium of Drosophila and mammalian kidneys. A relationship was noted between the disturbed balance of genetic material and congenital defects of the human excretory system.     

CELLULAR SPECIALIZATION IN THE EXCRETORY EPITHELIA OF AN INSECT, Macrosteles fascifrons STÅL (HOMOPTERA)

An electron microscopic investigation of the Malpighian tubules of a leaf hopper, Macrosteles fascifrons, shows that these organs comprise three quite distinct cell types, and the structure of these and of the mid- and hindgut epithelial cells is described. In particular, a comparison is made between the organization of the basal and apical surfaces of cells in the Malpighian tubule and in the vertebrate kidney, and it is suggested that similarities between these excretory epithelia reflect functional parallels between them. While the midgut and one region of the Malpighian tubule bear a typical microvillar brush border, elsewhere in the tubule and in the hindgut the apical surface bears cytoplasmic leaflets or lamellae. The sole solid excretory material of these insects consists of the brochosomes, secreted by cells of one region of the Malpighian tubule. The structure, geometry, and development of these unusual bodies, apparently formed within specialized Golgi regions, has been investigated, and histochemical tests indicate that they contain lipid and protein components.     

Ultrastructure of the malpighian tubules of Schistocerca gregaria

The ultrastructure of the Malpighian tubules of the adult desert locust, Schistocerca gregaria, is described. Male and female adults possess about 233 tubules, which empty proximally into the midgut-ileal region of the alimentary canal by way of 12 ampullae. The tubules vary from 10 mm to 23 mm in length. About one third of them are directed anteriorly, attaching distally at the caeca, while the remainder are directed posteriorly, attaching to other tubules, the rectum or large tracheal trunks adjacent to the hindgut. The Malpighian tubules from all locations examined consist of three ultrastructurally distinct regions: proximal, middle, and distal, referring to their position relative to the midgut. All cell types possess ultrastructural features characteristic of ion transporting tissue, i.e., elaboration of the basal and apical membranes and a close association of these membranes with mitochondria. The distal and proximal segments are short (1.5-1.7 mm) and heavily tracheated, and each is composed of a single, distinct cell type. The middle region is the longest segment of the Malpighian tubule and is composed of two distinct cell types, primary and secondary. Both cell types are binucleate. The more numerous primary cells have large nuclei, contain laminate concretions in membrane-bound vacuoles, and possess large microvilli that contain mitochondria. The secondary cells are smaller and possess smaller nuclei. The microvilli are reduced and lack mitochondria. Secondary cells do not contain laminate concretions. The possible compartmentalization of ion and fluid transport function based on segmentation in the Malpighian tubules is discussed.     

Regulation of Krüppel expression in the anlage of the Malpighian tubules in the Drosophila embryo

The expression of most Drosophila segmentation genes is not limited to the early blastoderm stage, when the segmental anlagen are determined. Rather, these genes are often expressed in a variety of organs and tissues at later stages of development. In contrast to the early expression, little is known about the regulatory interactions that govern the later expression patterns. Among other tissues, the central gap gene Krüppel is expressed and required in the anlage of the Malpighian tubules at the posterior terminus of the embryo. We have studied the interactions of Krüppel with other terminal genes. The gap genes tailless and huckebein, which repress Krüppel in the central segmentation domain, activate Krüppel expression in the posterior Malpighian tubule domain. The opposite effect on the posterior Krüppel expression is achieved by the interposition of another factor, the homeotic gene fork head, which is not involved in the control of the central domain. In addition, Krüppel activates different genes in the Malpighian tubules than in the central domain. Thus, both the regulation and the function of Krüppel in the Malpighian tubules differ strikingly from its role in segmentation.     

Characterization of tetraethylammonium uptake across the basolateral membrane of the Drosophila Malpighian (renal) tubule

Basolateral transport of the prototypical type I organic cation tetraethylammonium (TEA) by the Malpighian tubules of Drosophila melanogaster was studied using measurements of basolateral membrane potential (V(bl)) and uptake of [(14)C]-labeled TEA. TEA uptake was metabolically dependent and saturable (maximal rate of mediated TEA uptake by all potential transport processes, reflecting the total transport capacity of the membrane, 0.87 pmol.tubule(-1).min(-1); concentration of TEA at 0.5 of the maximal rate of TEA uptake value, 24 muM). TEA uptake in Malpighian tubules was inhibited by a number of type I (e.g., cimetidine, quinine, and TEA) and type II (e.g., verapamil) organic cations and was dependent on V(bl). TEA uptake was reduced in response to conditions that depolarized V(bl) (high-K(+) saline, Na(+)-free saline, NaCN) and increased in conditions that hyperpolarized V(bl) (low-K(+) saline). Addition of TEA to the saline bathing Malpighian tubules rapidly depolarized the V(bl), indicating that TEA uptake was electrogenic. Blockade of K(+) channels with Ba(2+) did not block effects of TEA on V(bl) or TEA uptake indicating that TEA uptake does not occur through K(+) channels. This is the first study to provide physiological evidence for an electrogenic carrier-mediated basolateral organic cation transport mechanism in insect Malpighian tubules. Our results also suggest that the mechanism of basolateral TEA uptake by Malpighian tubules is distinct from that found in vertebrate renal tubules.