Computed tomography with segmentation and quantification of individual organs in a D. melanogaster tumor model.

Drosophila melanogaster tumor models are growing in popularity, driven by the high degree of genetic as well as functional conservation to humans. The most common method to measure the effects of a tumor on distant organs of a human cancer patient is to use computed tomography (CT), often used in diagnosing cachexia, a debilitating cancer-induced syndrome most visibly characterized by loss of muscle mass. Successful application of high resolution micro-CT scanning of D. melanogaster was recently reported and we here present the segmentation of all visible larval organs at several stages of tumor development. We previously showed the strong expected reduction in muscle mass as the tumor develops, and we here report a surprisingly strong reduction also in gut and Malpighian tubules (kidney) volume. Time-point of tumor development was found to have a stronger correlation to cachectic organ volume loss than tumor volume, giving support to the previously proposed idea that tumor size does not directly determine degree of cachexia.

Buschke - Lowenstein anal tumor: an ambiguous entity.

The Buschke - Lowenstein tumor is a rare sexually transmitted disease. Its location at the anal margin is also very rare. The most incriminated risk factor is human papillomavirus infection. Its clinical form may be confusing with other tumor and infectious lesions. Histologically, it is characterized by a well-differentiated malpighian proliferation. It represents local aggressive behavior. The treatment of reference remains the surgery with healthy margins of excision. Other treatments have been tested, but their effectiveness remains uncertain. We report here a new case of anal margin Buschke - Lowenstein tumor with a review of the literature.

Endocrine regulation of MFS2 by branchless controls phosphate excretion and stone formation in Drosophila renal tubules.

How inorganic phosphate (Pi) homeostasis is regulated in Drosophila is currently unknown. We here identify MFS2 as a key Pi transporter in fly renal (Malpighian) tubules. Consistent with its role in Pi excretion, we found that dietary Pi induces MFS2 expression. This results in the formation of Malpighian calcium-Pi stones, while RNAi-mediated knockdown of MFS2 increases blood (hemolymph) Pi and decreases formation of Malpighian tubule stones in flies cultured on high Pi medium. Conversely, microinjection of adults with the phosphaturic human hormone fibroblast growth factor 23 (FGF23) induces tubule expression of MFS2 and decreases blood Pi. This action of FGF23 is blocked by genetic ablation of MFS2. Furthermore, genetic overexpression of the fly FGF branchless (bnl) in the tubules induces expression of MFS2 and increases Malpighian tubule stones suggesting that bnl is the endogenous phosphaturic hormone in adult flies. Finally, genetic ablation of MFS2 increased fly life span, suggesting that Malpighian tubule stones are a key element whereby high Pi diet reduces fly longevity previously reported by us. In conclusion, MFS2 mediates excretion of Pi in Drosophila, which is as in higher species under the hormonal control of FGF-signaling.

Toward a new insight of calcium oxalate stones in Drosophila by micro-computerized tomography.

We previously developed an animal model of calcium oxalate (CaOx) deposition on the Malphigian tubules of Drosophila melanogaster as a model of urolithiasis. Here, we introduce a new tool for the study of anatomical structure for Drosophila. As a consequence of technical development, the invention of micro-computerized tomography (CT) has been introduced to the small animal, such as rat and mice. We used Drosophila as a model organism and fed the flies 0.5% lithogenic agent ethylene glycol for 3 weeks. Samples were simply prepared for further scanned by micro-CT to scan samples at 800 nm resolution. CT scanning was performed at 40 kVp of voltage, 250 µA of current, and 1750 ms of exposure time and without filter. Reconstruction of sections was carried out with the GPU-based scanner software. Specific region of interests was further analyzed by DataViewer software. Area with high radiologic density level was defined as CaOx deposition for further 3D analysis. Image of whole lithogenic Drosophila was compared with control. High radiologic density level was detected in the region of Malphigian tubules which can be identified as CaOx stones. There was no stone image in the control group. The image was the same as human non-contrast CT for the diagnosis of stone disease. Micro-CT clearly demonstrated the calcium oxalate calcifications in the Malphigian tubules of fruit fly. The image system provides that a new vision on study animal will facilitate further study of stone disease. With the development of new technology on micro-CT, more delicate and advanced image will be presented in the future.

A Drosophila genetic model of nephrolithiasis: transcriptional changes in response to diet induced stone formation.

BACKGROUND: Urolithiasis is a significant healthcare issue but the pathophysiology of stone disease remains poorly understood. Drosophila Malpighian tubules were known to share similar physiological function to human renal tubules. We have used Drosophila as a genetic model to study the transcriptional response to stone formation secondary to dietary manipulation. METHODS: Wild-type male flies were raised on standard medium supplemented with lithogenic agents: control, sodium oxalate (NaOx) and ethylene glycol (EG). At 2 weeks, Malpighian tubules were dissected under polarized microscope to visualize crystals. The parallel group was dissected for RNA extraction and subsequent next-generation RNA sequencing. RESULTS: Crystal formation was visualized in 20%(±2.2) of flies on control diet, 73%(±3.6) on NaOx diet and 84%(±2.2) on EG diet. Differentially expressed genes were identified in flies fed with NaOx and EG diet comparing with the control group. Fifty-eight genes were differentially expressed (FDR <0.05, p < 0.05) in NaOx diet and 20 genes in EG diet. The molecular function of differentially expressed genes were assessed. Among these, Nervana 3, Eaat1 (Excitatory amino acid transporter 1), CG7912, CG5404, CG3036 worked as ion transmembrane transporters, which were possibly involved in stone pathogenesis. CONCLUSIONS: We have shown that by dietary modification, stone formation can be manipulated and visualized in Drosophila Malpighian tubules. This genetic model could be potentially used to identify the candidate genes that influence stone risk hence providing more insight to the pathogenesis of human stone disease.

Bicaudal C mutation causes myc and TOR pathway up-regulation and polycystic kidney disease-like phenotypes in Drosophila.

Progressive cystic kidney degeneration underlies diverse renal diseases, including the most common cause of kidney failure, autosomal dominant Polycystic Kidney Disease (PKD). Genetic analyses of patients and animal models have identified several key drivers of this disease. The precise molecular and cellular changes underlying cystogenesis remain, however, elusive. Drosophila mutants lacking the translational regulator Bicaudal C (BicC, the fly ortholog of vertebrate BICC1 implicated in renal cystogenesis) exhibited progressive cystic degeneration of the renal tubules (so called "Malpighian" tubules) and reduced renal function. The BicC protein was shown to bind to Drosophila (d-) myc mRNA in tubules. Elevation of d-Myc protein levels was a cause of tubular degeneration in BicC mutants. Activation of the Target of Rapamycin (TOR) kinase pathway, another common feature of PKD, was found in BicC mutant flies. Rapamycin administration substantially reduced the cystic phenotype in flies. We present new mechanistic insight on BicC function and propose that Drosophila may serve as a genetically tractable model for dissecting the evolutionarily-conserved molecular mechanisms of renal cystogenesis.

Expression of polyQ aggregates in Malpighian tubules leads to degeneration in Drosophila melanogaster.

BACKGROUND: Polyglutamine (polyQ) disorders are caused by expanded CAG (Glutamine) repeats in neurons in the brain. The expanded repeats are also expressed in the non-neuronal cells, however, their contribution to disease pathogenesis is not very well studied. In the present study, we have expressed a stretch of 127 Glutamine repeats in Malpighian tubules (MTs) of Drosophila melanogaster as these tissues do not undergo ecdysone induced histolysis during larval to pupal transition at metamorphosis. RESULTS: Progressive degeneration, which is the hallmark of neurodegeneration is also observed in MTs. The mutant protein forms inclusion bodies in the nucleus resulting in expansion of the nucleus and affect chromatin organization which appear loose and open, eventually resulting in DNA fragmentation and blebbing. A virtual absence of tubule lumen was observed followed by functional abnormalities. As development progressed, severe abnormalities affecting pupal epithelial morphogenesis processes were observed resulting in complete lethality. Distribution of heterogeneous RNA binding protein (hnRNP), HRB87F, Wnt/wingless and JNK signaling and expression of Relish was also found to be affected. Expression of resistance genes following polyQ expression was up regulated. CONCLUSION: The present study gives an insight into the effects of polyQ aggregates in non-neuronal tissues.

A Drosophila model identifies a critical role for zinc in mineralization for kidney stone disease.

Ectopic calcification is a driving force for a variety of diseases, including kidney stones and atherosclerosis, but initiating factors remain largely unknown. Given its importance in seemingly divergent disease processes, identifying fundamental principal actors for ectopic calcification may have broad translational significance. Here we establish a Drosophila melanogaster model for ectopic calcification by inhibiting xanthine dehydrogenase whose deficiency leads to kidney stones in humans and dogs. Micro X-ray absorption near edge spectroscopy (µXANES) synchrotron analyses revealed high enrichment of zinc in the Drosophila equivalent of kidney stones, which was also observed in human kidney stones and Randall's plaques (early calcifications seen in human kidneys thought to be the precursor for renal stones). To further test the role of zinc in driving mineralization, we inhibited zinc transporter genes in the ZnT family and observed suppression of Drosophila stone formation. Taken together, genetic, dietary, and pharmacologic interventions to lower zinc confirm a critical role for zinc in driving the process of heterogeneous nucleation that eventually leads to stone formation. Our findings open a novel perspective on the etiology of urinary stones and related diseases, which may lead to the identification of new preventive and therapeutic approaches.

Lamin-B in systemic inflammation, tissue homeostasis, and aging.

Gradual loss of tissue function (or homeostasis) is a natural process of aging and is believed to cause many age-associated diseases. In human epidemiology studies, the low-grade and chronic systemic inflammation in elderly has been correlated with the development of aging related pathologies. Although it is suspected that tissue decline is related to systemic inflammation, the cause and consequence of these aging phenomena are poorly understood. By studying the Drosophila fat body and gut, we have uncovered a mechanism by which lamin-B loss in the fat body upon aging induces age-associated systemic inflammation. This chronic inflammation results in the repression of gut local immune response, which in turn leads to the over-proliferation and mis-differentiation of the intestinal stem cells, thereby resulting in gut hyperplasia. Here we discuss the implications and remaining questions in light of our published findings and new observations.

Insect capa neuropeptides impact desiccation and cold tolerance.

The success of insects is linked to their impressive tolerance to environmental stress, but little is known about how such responses are mediated by the neuroendocrine system. Here we show that the capability (capa) neuropeptide gene is a desiccation- and cold stress-responsive gene in diverse dipteran species. Using targeted in vivo gene silencing, physiological manipulations, stress-tolerance assays, and rationally designed neuropeptide analogs, we demonstrate that the Drosophila melanogaster capa neuropeptide gene and its encoded peptides alter desiccation and cold tolerance. Knockdown of the capa gene increases desiccation tolerance but lengthens chill coma recovery time, and injection of capa peptide analogs can reverse both phenotypes. Immunohistochemical staining suggests that capa accumulates in the capa-expressing Va neurons during desiccation and nonlethal cold stress but is not released until recovery from each stress. Our results also suggest that regulation of cellular ion and water homeostasis mediated by capa peptide signaling in the insect Malpighian (renal) tubules is a key physiological mechanism during recovery from desiccation and cold stress. This work augments our understanding of how stress tolerance is mediated by neuroendocrine signaling and illustrates the use of rationally designed peptide analogs as agents for disrupting protective stress tolerance.

Ecdysone regulates morphogenesis and function of Malpighian tubules in Drosophila melanogaster through EcR-B2 isoform.

Malpighian tubules are the osmoregulatory and detoxifying organs of Drosophila and its proper development is critical for the survival of the organism. They are made up of two major cell types, the ectodermal principal cells and mesodermal stellate cells. The principal and stellate cells are structurally and physiologically distinct from each other, but coordinate together for production of isotonic fluid. Proper integration of these cells during the course of development is an important pre-requisite for the proper functioning of the tubules. We have conclusively determined an essential role of ecdysone hormone in the development and function of Malpighian tubules. Disruption of ecdysone signaling interferes with the organization of principal and stellate cells resulting in malformed tubules and early larval lethality. Abnormalities include reduction in the number of cells and the clustering of cells rather than their arrangement in characteristic wild type pattern. Organization of F-actin and ß-tubulin also show aberrant distribution pattern. Malformed tubules show reduced uric acid deposition and altered expression of Na(+)/K(+)-ATPase pump. B2 isoform of ecdysone receptor is critical for the development of Malpighian tubules and is expressed from early stages of its development.

Eliciting renal failure in mosquitoes with a small-molecule inhibitor of inward-rectifying potassium channels.

Mosquito-borne diseases such as malaria and dengue fever take a large toll on global health. The primary chemical agents used for controlling mosquitoes are insecticides that target the nervous system. However, the emergence of resistance in mosquito populations is reducing the efficacy of available insecticides. The development of new insecticides is therefore urgent. Here we show that VU573, a small-molecule inhibitor of mammalian inward-rectifying potassium (Kir) channels, inhibits a Kir channel cloned from the renal (Malpighian) tubules of Aedes aegypti (AeKir1). Injection of VU573 into the hemolymph of adult female mosquitoes (Ae. aegypti) disrupts the production and excretion of urine in a manner consistent with channel block of AeKir1 and renders the mosquitoes incapacitated (flightless or dead) within 24 hours. Moreover, the toxicity of VU573 in mosquitoes (Ae. aegypti) is exacerbated when hemolymph potassium levels are elevated, suggesting that Kir channels are essential for maintenance of whole-animal potassium homeostasis. Our study demonstrates that renal failure is a promising mechanism of action for killing mosquitoes, and motivates the discovery of selective small-molecule inhibitors of mosquito Kir channels for use as insecticides.

Drosophila TRPM channel is essential for the control of extracellular magnesium levels.

The TRPM group of cation channels plays diverse roles ranging from sensory signaling to Mg2+ homeostasis. In most metazoan organisms the TRPM subfamily is comprised of multiple members, including eight in humans. However, the Drosophila TRPM subfamily is unusual in that it consists of a single member. Currently, the functional requirements for this channel have not been reported. Here, we found that the Drosophila TRPM protein was expressed in the fly counterpart of mammalian kidneys, the Malpighian tubules, which function in the removal of electrolytes and toxic components from the hemolymph. We generated mutations in trpm and found that this resulted in shortening of the Malpighian tubules. In contrast to all other Drosophila trp mutations, loss of trpm was essential for viability, as trpm mutations resulted in pupal lethality. Supplementation of the diet with a high concentration of Mg2+ exacerbated the phenotype, resulting in growth arrest during the larval period. Feeding high Mg2+ also resulted in elevated Mg2+ in the hemolymph, but had relatively little effect on cellular Mg2+. We conclude that loss of Drosophila trpm leads to hypermagnesemia due to a defect in removal of Mg2+ from the hemolymph. These data provide the first evidence for a role for a Drosophila TRP channel in Mg2+ homeostasis, and underscore a broad and evolutionarily conserved role for TRPM channels in Mg2+ homeostasis.

Structural and ultrastructural alterations of Malpighian tubules of Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) larvae infected with different Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) recombinant viruses.

Malpighian tubules constitute the main excretion organ of insects. Infection by egt(-) recombinant AcMNPV baculovirus in lepidopteran larvae promotes early degeneration of these structures, which has been correlated with earlier death of the host. However, no trace of viral infection has been detected in that tissue. We constructed two AgMNPV recombinants with the egfp gene under control of the hsp70 promoter, one being egt(-), and used another two recombinants (one egt(-)) containing the lacZ gene. Morphological alterations in the tubules were analyzed by light and electron microscopies. Bioassays were conducted to compare the pathogenicity of recombinants. Results showed progressive presence of marker proteins and tissue degeneration without signals of infection in the tissue. Morphological and bioassay results showed increased pathogenicity for lacZ-containing recombinants compared to the egfp ones; as for egt(-) viruses, we noted higher intensity and earlier onset of alterations. The absence of infection led us to believe that Malpighian tubules degeneration is provoked initially by the death of tracheal cells attached to the tubules and later, by the death of Malpighian tubule cells themselves. Tubule cell death might be due to oncosis and apoptosis, which may be activated by depletion of energy reserves and by accumulation of marker proteins, respectively. Absence of the egt gene may be leading to a higher energetic expense due to molting, thus aggravating tubule cell death, resulting in faster death of host.

Genome-wide survey of V-ATPase genes in Drosophila reveals a conserved renal phenotype for lethal alleles.

V-ATPases are ubiquitous, vital proton pumps that play a multiplicity of roles in higher organisms. In many epithelia, they are the major energizer of cotransport processes and have been implicated in functions as diverse as fluid secretion and longevity. The first animal knockout of a V-ATPase was identified in Drosophila, and its recessive lethality demonstrated the essential nature of V-ATPases. This article surveys the entire V-ATPase gene family in Drosophila, both experimentally and in silico. Adult expression patterns of most of the genes are shown experimentally for the first time, using in situ hybridization or reporter gene expression, and these results are reconciled with published expression and microarray data. For each subunit, the single gene identified previously by microarray, as upregulated and abundant in tubules, is shown to be similarly abundant in other epithelia in which V-ATPases are known to be important; there thus appears to be a single dominant "plasma membrane" V-ATPase holoenzyme in Drosophila. This provides the most comprehensive view of V-ATPase expression yet in a multicellular organism. The transparent Malpighian tubule phenotype first identified in lethal alleles of vha55, the gene encoding the B-subunit, is shown to be general to those plasma membrane V-ATPase subunits for which lethal alleles are available, and to be caused by failure to accumulate uric acid crystals. These results coincide with the expression view of the gene family, in which 13 of the genes are specialized for epithelial roles, whereas others have spatially or temporally restricted patterns of expression.

Role of habitat components on the dynamics of Aedes albopictus (Diptera: Culicidae) from New Orleans.

Monthly sampling of tire pile populations of Aedes albopictus (Skuse) in Orleans Parish, New Orleans, LA, was done in 1995 to determine prevalence of ascogregarine parasites and changes in wing length. Prevalence of Ascogregarina taiwanensis (Lien & Levine) infection was 100% in midsummer and decreased in the fall and spring (60-70%). Wing lengths were longest in the spring and fall and shortest in midsummer. We evaluated the effect of A. taiwanensis infections under high and deficient levels of leaf litter nutrients on mortality, development time, wing length, and reproductive potential of a New Orleans strain of Ae. albopictus. Parasitism and deficient nutrients caused a 35% increase in the rate of larval mortality and significantly extended the development time of females. Parasitized adults were 5% smaller and produced 23% fewer eggs than unparasitized siblings. In addition, abnormal Malpighian tubule morphology and melanization of ascogregarines were seen in adults from nutrient-deficient microcosms. We conclude that ascogregarine infections affect the dynamics of Ae. albopictus by increasing the mortality of immature stages when nutrients supplies are scarce, and by decreasing the reproductive capacity of females under high nutrient conditions.

The products of ribbon and raw are necessary for proper cell shape and cellular localization of nonmuscle myosin in Drosophila.

Mutations in the genes rib and raw cause defects in the morphology of a number of tissues in homozygous mutant embryos. A variety of tubular epithelial tissues adopt a wide, round shape in mutants and dorsal closure fails. Cells of the normal tubular epithelia are columnar and wedge-shaped, and cells of the epidermis become elongated dorsoventrally as dorsal closure occurs. However, the cells of mutants are round or cuboidal in all of the tissues with mutant phenotypes, consistent with the hypothesis that the products of these genes are required for proper cell shape. Cytoskeletal defects, in particular, defects in myosin-driven contraction of the cortical actin cytoskeleton, could be responsible for the lack of specific cell shapes in mutant embryos. This possibility is supported by our observation that the intracellular localization of nonmuscle myosin to the leading edge of the dorsally closing epidermis is absent or reduced in rib and raw mutant embryos. In contrast, the band of actin that is also located at the leading edge is neither eliminated nor interrupted by either rib or raw mutations. Furthermore, mutations of zipper, the gene encoding the nonmuscle myosin heavy chain, exhibit mutant phenotypes in most of the same tissues affected by rib and raw, and many of the phenotypes are similar to those of rib and raw. Therefore, the products of rib and raw may be required for proper myosin-driven contraction of the actin cytoskeleton.

Cold inhibition of cell volume regulation during the freezing of insect malpighian tubules.

Cells in freeze-tolerant tissues must survive substantial shrinkage during exposure to the hyperosmolarity that results as solutes are excluded from extracellular ice. We investigated the possibility that this hyperosmotic shock elicits an acute regulatory volume increase (RVI) by monitoring the response of epithelial cell volume in the Malpighian tubules of the New Zealand alpine weta (Hemideina maori) during exposure to low temperature/hyperosmolarity (mimicking freezing conditions) or during an actual freeze/thaw cycle. The cross-sectional area of cells in isolated Malpighian tubules was measured using differential interference contrast microscopy. At 20 degrees C, cells held in saline containing 400 mmol1-1 glucose exhibit an RVI in response to hyperosmotic shock. Cross-sectional area decreased by 30% immediately after a change from iso-osmotic (0.7 osmol1-1) to hyper-osmotic saline (2.1 osmol1-1, equal to the osmotic shock encountered during freezing to -4 degrees C) and then returned to 21% below the control value 30 min after the exposure. Although substantial cellular function of Malpighian tubules was retained at low temperature (the rate of fluid secretion by isolated tubules at 4 degrees C was 72% of that measured at 20 degrees C), no RVI was observed at 0% degrees C; cross-sectional area was 39% below the control value immediately after the hyperosmotic exposure and 36% below the control value 30 min after hyperosmotic exposure. Dibutyryl cyclic AMP potentiated the RVI observed at 20 degrees C, but failed to elicit an RVI at 0 degrees C. A substantial RVI was also absent when the saline contained trehalose rather than glucose, regardless of whether the tubules were held at 20 degrees C or 0 degrees C. The cross-sectional area of cells in saline containing glucose remained at approximately 30% below the control value during an entire 30 min period of actual freezing to -4 degrees C, suggesting that an acute volume regulatory response was in fact inhibited during mild freezing. The inhibition of an acute RVI during mild freezing may serve to avoid the energetic expenditure associated with volume regulation at a time when the normal defence of cell volume appears to be unnecessary.

Quantitative assessment of cell damage in situ: electron microprobe X-ray analysis of model organisms treated with noxious species.

The existing set of methods for assessing toxicity of noxas, based on experiments with whole animals (subclinical toxicity, toxicokinetics, carcinogenity, teratogenity, neurotoxicology etc.) does not provide much information about cellular and subcellular effects such compounds may exert. We suggest to complement the current methodology by combining a traditional morphological observation in an electron microscope with a spectroscopic method of electron microprobe X-ray analysis (or X-ray microanalysis). The latter makes it possible to measure concentrations of chemical elements in individual cells and organelles and effects of noxas can thus be assessed (i) at subcellular level, (ii) directly in situ and (iii) quantitatively. Concentrations of biologically important elements such as phosphorus, sulfur or zinc were measured in individual organelles in both intact and noxa-treated tissues, thus offering a possibility of comparing the effects of various noxious species at subcellular level (with the noxa previously applied to whole tissue or animal). The suggested correlation of analytical and morphological information may also provide new insights into cellular targeting of noxas (and potentially also drugs) as some organelles appear to be much more susceptible to damage than others.

Deletion of the baculovirus ecdysteroid UDP-glucosyltransferase gene induces early degeneration of Malpighian tubules in infected insects.

Deletion of the ecdysteroid UDP-glucosyltransferase gene (egt) from the Autographa californica nuclear polyhedrosis virus (AcNPV) genome increases the speed of killing of this virus (D. R. O'Reilly and L. K. Miller, Bio/Technology 9:1086-1089, 1991). Second-instar Spodoptera exigua larvae are killed more rapidly by the egt deletion mutant of AcNPV than by wild-type AcNPV. Unlike wild-type AcNPV-infected larvae, larvae infected with an egt deletion mutant molt and resume feeding as mock-infected larvae do. Wild-type AcNPV and egt deletion mutant recombinants marked with a lacZ gene were used to study their pathogenesis in insects. Histopathological investigation revealed that early degeneration of the Malpighian tubules, not the molting per se, may be the cause of this increased speed of killing by AcNPV.