Effects of bafilomycin A1 and amiloride on the apical potassium and proton gradients in Drosophila Malpighian tubules studied by X-ray microanalysis and microelectrode measurements

The intracellular distribution of potassium in Malpighian tubules from Drosophila larva was measured by electron probe X-ray microanalysis of freeze-dried cryosections. Application of amiloride alone to the haemolymph space had no effect on the intracellular potassium concentration in the region of intermediate cytoplasm (between the basal region of basal membrane infoldings and the apical brush border), whereas a potassium increase as well as a chloride increase was observed after simultaneous blocking of the potassium conductance of the basal membrane with barium. Injected bafilomycin and amiloride applied in the haemolymph caused an increase of the potassium content in the basal cytoplasm but not in the microvilli. In addition, the intracellular water portion was decreased by bafilomycin. pH measurements in isolated larval anterior tubules with proton-selective microelectrodes showed that bafilomycin added to the bathing solution caused a decrease in intracellular pH. Addition of amiloride had no significant effect on intracellular pH, but the pH of the luminal fluid was decreased within 1 min by 0.5 pH units. The amiloride-induced luminal pH decrease could be inhibited by the metabolic blocker KCN as well as by bafilomycin. Furthermore, removing potassium from the bathing saline caused a slow luminal acidification, which could be blocked by KCN. Our results support the hypothesis of a functionally coupled transport system in the apical membrane consisting of a bafilomycin-sensitive V-ATPase and a K⁺-dependent, amiloride-sensitive K⁺/H⁺ exchange system.Abbreviation C _a element concentration related to water - C _d element content related to dry weight - dw dry weight - DMSO dimethylsulphoxide - emf electromotive force - NBD-Cl 7-chloro-4-nitrobenz-2-oxa-1,3-diazole - NEM N-ethylmaleimide - NMDG⁺ N-methyl-d-glucamine - PD potential difference - pH_i intracellular pH value - pH_lu luminal pH value - pmf protonmotive force - SD standard deviation - SE standard error - STEM scanning transmission electron microscopy - V _a apical potential difference - V _b basal potential difference - V _t transepithelial potential difference     

Intracellular pH regulation by the plasma membrane V-ATPase in Malpighian tubules of Drosophila larvae

The functional significance of the apical vacuolar-type proton pump (V-ATPase) in Drosophila Malpighian tubules was studied by measuring the intracellular pH (pH_i) and luminal pH (pH_lu) with double-barrelled pH-microelectrodes in proximal segments of the larval anterior tubule immersed in nominally bicarbonate-free solutions (pH_o 6.9). In proximal segments both pH_i (7.43±0.20) and pH_lu (7.10±0.24) were significantly lower than in distal segments (pH_i 7.70±0.29, pH_lu 8.09±0.15). Steady-state pH_i of proximal segments was much less sensitive to changes in pH_o than pH of the luminal fluid (pH_lu/pH_o was 0.49 while pH_i/pH_o was 0.18; pH_o 6.50–7.20). Re-alkaliniziation from an NH₄Cl-induced intracellular acid load (initial pH_i recovery rate 0.55±0.34 pH·min^-1) was nearly totally inhibited by 1 mmol·l^-1 KCN (96% inhibition) and to a large degree (79%) by 1 mol·l^-1 bafilomycin A₁. In contrast, both vanadate (1 mmol·l^-1) and amiloride (1 mmol·l^-1) inhibited pH_i recovery by 38% and 33%, respectively. Unlike amiloride, removal of Na⁺ from the bathing saline had no effect on pH_i recovery, indicating that a Na⁺/H⁺ exchange is not significantly involved in pH_i regulation. Instead pH_i regulation apparently depended largely on the availability of ATP and on the activity of the bafilomycin-sensitive proton pump.Abbreviations DMSO dimethylsulphoxide - DNP 2,4-dinitrophenol - NMDG N-methyl-D-glucamine - pH_i intracellular pH - pH_lu pH of the luminal fluid - pH_o pH of the superfusion medium - _I intrinsic intracellular buffer capacity     

Genetic Control of Development of Malpighian Tubules 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 ofDrosophila and mammalian kidneys. A relationship was noted between the disturbed balance of genetic material and congenital defects of the human excretory system.     

Purine transport by Malpighian tubules of pteridine-deficient eye color mutants of Drosophila melanogaster

Uptakes of guanine into Malpighian tubules of wild-type Drosophila and the eye color mutants white (w), brown (bw), and pink-peach (p ^p) have been compared. Tubules for each of these mutants are unable to concentrate guanine intracellularly. The transport of xanthine and riboflavin is also deficient in w tubules. The transport of guanosine, adenine, hypoxanthine, and guanosine monophosphate is similar in wild-type and white Malpighian tubules. These data and other information about these mutants make it likely that these pteridine-deficient eye color mutants do not produce pigments because of the inability to transport a pteridine precursor. This view supports the hypothesis that mutants which lack both pteridine and ommochromes do so because precursors to both classes of pigments share a common transport system.This work was supported by Grant GM22366 from NIH.     

Partial hyperploidy of the X-chromosome inDrosophila hydei leading to duplication of male gonadal and genital structures

Summary Introduction of two doses of the X-chromosomalw^mCo duplication next to a normal X-chromosome in males ofD. hydei leads to duplication of testis tissue and structures derived from the male genital disc. The effect of this partial hyperploidy of the X-chromosome seems restricted to the male. We tentatively conclude that this part of the X-chromosome contains some factor(s) which may specifically affect the reproductive system and analia of males.     

Anti-actin immunoreactivity is retained in rhabdoms of Drosophila ninaC photoreceptors

Summary The Drosophila ninaC mutation produces small rhabdomeres with the axial filament of the microvillar cytoskeleton reduced or missing. Using post-embedding immunogold labelling of LR White-embedded eyes, we show that several alleles of this mutation retain positive anti-actin immunoreactivity in the rhabdomeres, comparable to that of wild-type flies.     

Temporal control of urate oxidase activity during development of the third-instar larva of Drosophila: The role of 20-hydroxyecdysone

The tissue-specific enzyme urate oxidase is confined exclusively to the Malpighian tubules of Drosophila melanogaster and expressed only in the third-instar larva and the adult. Shortly before pupariation urate oxidase activity declines precipitously and is not detectable 24 hours later. That 20-hydroxyecdysone is the factor that triggers the disappearance of urate oxidase activity in late third-instar larvae is demonstrated using the temperature sensitive mutant ecd¹ which at the nonpermissive temperature of 29°C fails to accumulate a sufficient concentration of 20-hydroxyecdysone necessary for puparium formation and thus remains a third-instar larva for 1 to 2 weeks before death. Both the life cycle and the temporal profile of urate oxidase activity in ecd¹ larvae at 19°C is identical to that of the wild type. However, at 29°C ecd¹ third-instar larvae retain high urate oxidase activity. A precipitous decline in urate oxidase activity is observed when ecd¹ larvae at 29°C are fed 20-hydroxyecdysone. These data implicate 20-hydroxyecdysone in the process that controls the rapid decline of urate oxidase activity at the time of puparium formation. In whole homogenates of Malpighian tubules, the urate oxidase polypeptide was identified in SDS-polyacrylamide gels by its Rf with respect to homogeneously pure Drosophila urate oxidase and also by immunoprecipitation with rabbit anti-Drosophila urate oxidase IgG. Throughout development the amount of the urate oxidase polypeptide is correlated with the magnitude of urate oxidase activity.     

Segmental peptidergic innervation of abdominal targets in larval and adult dipteran insects revealed with an antiserum against leucokinin I

Summary An antiserum against the cockroach neuropeptide leucokinin I (LKI) was used to study peptidergic neurons and their innervation patterns in larvae and adults of three species of higher dipteran insects, the flies Drosophila melanogaster, Calliphora vomitoria, and Phormia terraenovae, as well as larvae of a primitive dipteran insect, the crane fly Phalacrocera replicata. In the larvae of the higher dipteran flies, the antiserum revealed three pairs of cells in the brain, three pairs of ventro-medial cells in the subesophageal ganglion, and seven pairs of ventro-lateral cells in the abdominal ganglia. Each of these 14 abdominal leucokinin-immunoreactive (LKIR) neurons innervates a single muscle of the abdominal body wall (muscle 8), which is known to degenerate shortly after adult emergence. Conventional electron microscopy demonstrates that this muscle is innervated by at least one axon containing clear vesicles and two axons containing dense-cored vesicles. Electronmicroscopical immunocytochemistry shows that the LKIR axon is one of these two axons with dense-cored vesicles and that it forms terminals on the sarcolemma of its target muscle. The abdominal LKIR neurons appear to survive metamorphosis. In the adult fly, the efferent abdominal LKIR neurons innervate the spiracles, the heart, and neurohemal regions of the abdominal wall. In the crane fly larva, dorso-medial and ventrolateral LKIR cell bodies are located in both thoracic and abdominal ganglia of the ventral nerve cord. As in the larvae of the other flies, the abdominal ventrolateral LKIR neurons form efferent axons. However, in the crane fly larva there are two pairs of efferent LKIR neurons in each of the abdominal ganglia and their peripheral targets include neurohemal regions of the dorsal transverse nerves. An additional difference is that in the crane fly, a caudal pair of LKIR axons originating from the penultimate pair of dorso-median LKIR cells in the terminal ganglion innervate the hindgut.     

Established cell lines ofDrosophila hydei

Summary Four cell lines have been isolated fromDrosophila hydei embryos. Three lines have a normal XY karyotype, the fourth has an XO karyotype with an additional small heterochromatic fragment. The cells contain presumable cytoplasmic virus like particles. This work was supported by a travel grant to Dr. N. H. Lubsen from the Netherlands Organization for the Advancement of Pure Research (Z. W. O.).     

Bristle patterns and clones along a compartment border in the anterior wing margin ofDrosophila hydei

Summary The bristle pattern along the first longitudinal vein of the wing ofD. hydei differs from that ofD. melanogaster. Instead of a triple row,D. hydei and some allied species show a pattern of five parallel bristle rows of which the medial row (MR) is comparable to the medial triple row (MTR) ofD. melanogaster. Cells of the MR can be made homozygousyellow (y) by induction of mitotic recombination in heterozygousy/y⁺ females. Until 70 h after egg laying (AEL), the MR clones inD. hydei overlap with one or more of the accompanying dorsal and ventral bristle rows. Between 70 and 120 h AEL the MR clones only overlap with dorsal bristle rows. Some time later they also become separated from both dorsal rows. The resulting MR clone pattern fits with the overall longitudinal clone pattern in the wing blade ofD. melanogaster described by Bryant (1970) and others. The MR clones inD. hydei, however, often show a fragmented appearance with many indentations of the surroundingy⁺ tissue even when induced after fixation of the DV compartment boundary. This result contrasts with the commonly held notion, derived from work withD. melanogaster, that compartment boundaries are smooth lines.     

Presence of Locusta diuretic hormone in endocrine cells of the ampullae of locust Malpighian tubules

This is an investigation of an endocrine cell type in the midgut of the migratory locust Locusta migratoria. This cell type is found in the posterior region of the midgut and is especially common in the ampullae through which Malpighian tubules drain into the gut at the midgut-hindgut junction. Strong Locusta diuretic hormone-like immunoreactivity in these cells was colocalized with FMRFamide- and substance P-like immunoreactivities. At the ultrastructural level, immunoreactivity for Locusta diuretic hormone was found in spherical granules (mean diameter of 450 nm), the contents of which showed variable electron density. Fractionation of a methanolic extract of the ampullae by reversed-phase high performance liquid chromatography revealed the presence of two peaks of Locusta diuretic hormone-like immunoreactive material, both of which stimulate cyclic AMP production by isolated Malpighian tubules. The more hydrophobic material is most likely Locusta diuretic hormone, which has the same retention time when chromatographed under identical conditions. Received: 15 September 1995 / Accepted: 16 February 1996     

Isolation and characterization of gap junctions from Drosophila melanogaster

Summary A procedure has been developed to isolate gap junction-enriched subcellular fractions from Drosophila. Crude membranes from larval homogenates were extracted with 1% N-lauroyl sarcosine in 6 M urea and the gap junctions were collected by centrifugation. The major proteins were separated by SDS PAGE and purified by electro-elution. Electron microscopy revealed structurally pleiomorphic gap junctions in the fractions which included (1) conventional, 16–18 nm-wide septalaminar, (2) collapsed, 13–15 nm-wide pentalaminar, (3) split, and (4) aggregated forms. The fractions contained five major proteins with apparent molecular weights of 18, 26, 36, 52 and 54 kD. Evidence based on (1) the degradation and aggregation behavior of the major proteins following electro-elution and reelectrophoresis, (2) immunological cross-reactivities by affinity-purified antibodies against the major proteins on immunoblots, and (3) immunofluorescent staining of presumptive gap junctions in Drosophila imaginal discs at the light-microscopic level and immunogold staining of purified gap junctions at the electron-microscopic level suggests that the major proteins are interrelated and of gap-junction origin.     

The Cost of Being Bright

Male ornaments are predicted to be a signal of the genetic quality of a male but often the relationship between ornaments and the physiological performances of males is unclear. Males of Metichnogaster cillipennis, a hover-wasp, during their courtship flights send luminous signals to females by using two abdominal reflectors. Malpighian tubules, an essential organ in the physiology of insects, participate in the building of the larger of these reflectors but at a physiological cost. By evaluating the capacity of light reflection from males during their courtship, females can assess directly the physiological quality of males.     

Stimulus-permeability coupling in rat pulmonary macrophages challenged by Pseudomonas aeruginosa

Summary Electron probe X-ray microanalysis (XRMA) of freeze-dried ultrathin sections provides the capability of measuring intracellular elemental content. This methodology was used to investigate the stimulus-permeability coupling responses associated with phagocytosis of Pseudomonas aeruginosa by cultured pulmonary alveolar macrophages (PAMs) of rats. PAMs were challenged with P. aeruginosa suspended in Gey's buffer at a bacteria to PAM ratio of 501 for 1 h at 37° C. A 1-mm³ pellet of the unchallenged control PAMs, challenged PAMs and P. aeruginosa alone was quench-frozen in nitrogen-cooled, liquid propane, and 0.1-m cryosections were cut at -100° C. X-ray spectra were collected for nucleus and cytoplasm of 39 control PAMs, 36 challenged PAMs and 40 P. aeruginosa. Concentrations (mmole/kg dry weight) were obtained for Na, Cl, K, Ca, Mg, P, S for each cell. In the control PAMs, the content was similar to other mammalian cells. Moreover, there were no differences in elemental content between nucleus and cytoplasm. In the challenged PAMs, Na concentration was 4 times that of control PAMs (p<0.001) whereas Cl was double (p<0.001), K was 29% lower (p<0.001), and Ca was 4 times higher (p<0.05). The elemental concentration profile in the P. aeruginosa was distinctly different from that of the PAMs: higher Na, Ca, Mg, but lower Cl and K values. These results demonstrate elemental content changes in cultured PAMs challenged with P. aeruginosa that indicate a stimulus-permeability response by membranes associated with the phagocytic process.     

Insectivory and social digestion inDrosophila

It has long been believed thatDrosophila larvae feed almost entirely by ingesting yeast and possibly other microorganisms that are associated with fermenting fruits or other vegetable matter. However, we have discovered that the larvae of a number ofDrosophila species can consume such diverse substrates as insect tissues, including the exoskeleton. Experiments reported here, which include raising sterile dechorionated eggs to adulthood on adult carcasses under axenic conditions, show that larvae can consume complex chitinous substrates directly without the assistance of microorganisms. We show thatDrosophila larvae are able externally to digest amylose, cellulose, and chitin, without coming into physical contact with them. We conclude that not only doDrosophila larvae produce enzymes enabling them to digest a wide variety of substrates, but also these enzymes are egested onto the substrates so that at least some digestion, especially of large polymers, takes place externally. Finally, we suggest that the phenomenon of external digestion explains both the previously unexplained massiveness ofDrosophila salivary glands and their chromosomes and the tendency of larvae to cluster, which may also be true of other dipterans.This investigation was supported by NIH (AREA) Grant IR 15AG05897.