Fluid and cation secretion by the Malpighian tubules of Locusta

In vitro preparations of Locusta Malpighian tubules are able to transport K⁺ against its concentration gradient. The ‘urine’ is slightly hyper-osmotic with respect to the bathing solution and the rate of secretion is inversely dependent on the osmotic pressure of the latter. The rate of fluid secretion increases with increasing temperature; being maximal at approx 40°C. The ionic composition of the secreted fluid, as indicated by Na⁺/K⁺ ratios, is altered by the presence of 1 mM ouabain in the bathing solution. Fluid secretion is inhibited by 1 mM ouabain. In addition, oxygen consumption by the Malpighian tubules is inhibited by either the presence of 1 mM ouabain or the absence of K⁺ in the bathing solution. The relationship between respiration, active transport and the Na⁺K⁺-activated ATPase is discussed.     

Ultrastructural evidence for paracellular fluid flow in the Malpighian tubules of a larval mayfly

The ultrastructure of the Malpighian tubules of larvae of the Mayfly Ecdyonurus dispar (Ephemeroptera) is described. There are about 60 tubules, which consist of four distinct regions. The most proximal section (region I) appears to be responsible for fluid secretion. A unique feature is the presence of channels leading off the main lumen, which end close to the basal border of the cells. Microvilli are confined to these channels in region I. Region II is a short spiral region, the cells of which possess long basal folds and associated mitochondria. Region III is a simple conducting tube leading to one of six collecting ducts (region IV) arranged radially around the gut. In each collecting duct there are two cell types present. Type 2 cells are relatively simple, but give rise to numerous, long, microvilli-like projections. Type 1 cells possess long basal folds, and curious membrane whorls in the apical zone. Evidence is presented which suggest that water movements into region I takes place via the paracellular route. Region II is probably a reabsorptive region, but the function of region IV, based on ultrastructural evidence is more difficult to elucidate.     

Effects of biogenic amines and hormones on butterfly Malpighian tubules: Dopamine stimulates fluid secretion

Isolated Malpighian tubules of Papilio demodocus, the citrus swallowtail butterfly, were stimulated by biogenic amines as well as by cyclic AMP and the naturally occuring diuretic hormone. The greatest secretory response was obtained with 5-hydroxytryptamine, and smaller responses with dopamine and noradrenaline, but none of these amines could induce the maximal secretion rates obtained with cyclic AMP and diuretic hormone. Various other biogenic amines, hormones and pharmacological agents, including adrenaline, had no effect on Papilio tubules. The blocking agents cyproheptadine, phentolamine and propranolol did not alter the tubule response to biogenic amines.     

Effects of anions, acetazolamide, thiocyanate and amiloride on fluid secretion by the Malpighian tubules of Locusta migratoria L.

The effect of Cl⁻, HCO₃⁻, Br⁻, acetazolamide, thiocyanate and amiloride on urine formation in Locusta migratoria Malpighian tubules have been determined. The rate of fluid secretion depends markedly on the concentration of Cl⁻ in the bathing solution with concentrations less than 90 mM resulting in reduced fluid secretion. Substitution of Br⁻ for Cl⁻ had no significant effect on the rate of the fluid secretion. Replacement of NaHCO₃ with NaCl in Hepes buffered Ringer solution reduced the rate of urine production by 23%. Fluid secretions were reduced in the presence of 10⁻⁴–10⁻² M acetazolamide, a carbonic anhydrase inhibitor. The combined effect of acetazolamide in the absence of HCO₃⁻ appears to be additive. A 1 mM concentration of thiocyanate, an ionic inhibitor, reduced fluid secretion by 35%. Amiloride interferes with the electrogenic entry of Na⁺ into the cell and a 1 mM solution reduced fluid secretion by 94% with secretion completely inhibited in 80% of the tubules tested.     

Stimulatory and inhibitory effects of endogenous factors in head extracts of Formica polyctena (Hymenoptera) on the fluid secretion of Malpighian tubules

This study was designed to investigate the regulation of fluid secretion by the Malpighian tubules of the worker ant Formica polyctena (Hymenoptera). Different solvent systems were used to make crude head extracts and to determine the solubility of the diuretic factors. Surprisingly, when distilled water, acid acetone, methanol and 15% trifluoroacetic acid (TFA) were used as solvents, two consecutive significant stimulations of fluid secretion were obtained: the first, when adding the extract to the tubule and the second, when washing it out. Extract obtained with a fifth solvent, Ringer solution, gave an almost complete but reversible inhibition of fluid secretion. Extracts were prepurified by means of a disposable C₁₈ column by elution with 20, 40, 60 and 80% acetonitrile. When the fractions were kept apart the 40% acetonitrile fraction caused an inhibition of fluid secretion. The 20, 60 and 80% acetonitrile fractions on the other hand resulted in two consecutive stimulations as described above. The dose-response curve for 15% TFA extract was bell-shaped with a threshold concentration of 1 × 10⁻³ heads/μl Ringer. A maximum response (stimulation of fluid secretion by a factor of 3.3 ± 0.72, n = 10) was observed with a concentration of 5 × 10⁻² heads/μl Ringer. Higher concentrations resulted in small increases of fluid secretion rates and in the appearance of the second stimulation when the extract was washed out. The activity present in the heads of Formica was not destroyed by boiling or by proteolytic enzymes (trypsin, chymotrypsin, pronase E and proteinase K). Only immobilized aminopeptidase M, which destroys the activity of peptides with a free N-terminus, had a significant effect on the activity of a 15% TFA head extract. Various biogenic amines were tested for their ability to mimic the effect of the head extracts. Only octopamine and dopamine evoked a small and transient increase in secretion rate. Thus biogenic amines probably do not contribute to a large extent to the response of Formica tubules to the crude head extract. The possibility that both diuretic and antidiuretic factors are present in the extract is discussed.     

Stimulation of fluid secretion by single isolated Malpighian tubules of the house cricket, Acheta domesticus

Abstract. Fluid secretion by single isolated Malpighian tubules of Acheta domesticus (L.) is stimulated by aqueous extracts of nervous and neuroendocrine tissues from the cricket. Diuretic activity, expressed as the increase in rate of secretion per microgram tissue protein (pl/mm/min/ug protein), is highest in the CA and CC.
The response to aqueous CC extracts is dose-dependent. The maximum increase in secretion rate is 300–350 pl/mm/min and the ED_max and ED₅₀ are 0.1–0.2 and 0.02–0.03 gland pairs respectively.
The diuretic activity in the CC is retained after 5min at 95C, and is freely soluble in 80% methanol. Diuretic activity is, however, greatly reduced after prolonged heating or after treatment with either pronase or chymotrypsin. It is conluded that the diuretic factor(s) is a low molecular weight peptide. A loss of activity after incubation with pyroglutamate amino peptidase suggests that some active peptides present are N-terminally blocked.
The diuretic activity of crude aqueous CC extracts is rapidly lost on standing at room temperature. This is partly prevented by precipitation of protein enzymes by either heat treatment or extraction in methanol.
Synthetic vertebrate and insect neuropeptides are generally low in activity increasing fluid secretion by no more than 60pl/mm/min. However, extracts of neuroendocrine tissues from a wide range of insect species are potent stimulants of tubule secretion. Various biogenic amines were tested and have little effect on fluid secretion; thus they cannot contribute greatly to the response of cricket tubules to aqueous tissue extracts.     

Invitro effect of Canavalia ensiformis urease and the derived peptide Jaburetox-2Ec on Rhodnius prolixus Malpighian tubules

Ureases are metalloenzymes that are widespread among plants, fungi and bacteria. Urease isoforms (jack bean urease-JBU and canatoxin) from Canavalia ensiformis seeds are toxic to insects and fungi, suggesting a role in plant defense. The entomotoxic effect is due to the release of a 10-kDa peptide by cathepsin-like enzymes in the insect's midgut. Urease causes a decrease in post-feeding weight loss in Rhodnius prolixus, suggesting an effect on water balance. To investigate how this impairment occurs, we have evaluated the action of JBU and the urease-derivated peptide Jaburetox-2Ec on R. prolixus Malpighian tubules and also investigated the involvement of second messengers. JBU and Jaburetox-2Ec affect serotonin-induced secretion from Malpighian tubules. This effect is not cAMP-dependent, but the Jaburetox-2Ec effect is cGMP-dependent. Eicosanoid metabolites and calcium ions appear to be involved in JBU effect on diuresis, but are not involved in the action of Jaburetox-2Ec. Jaburetox-2Ec, but not JBU, causes a change in the transepithelial potential of the tubules. Canatoxin has a similar effect on tubules secretion, decreasing the secretion rate, but the urease from Helicobacter pylori has no significant effect. These data are helpful in our understanding of the actions of ureases and derived peptides on insects, and also reinforces the potential use of these proteins as biopesticides.     

Effect of Na+, K+, ouabain,amiloride and ethacrynic acid on the transepithelial potential across Malpighian tubules of Locusta

A transepithelial potential of +8.74±0.29 mV (n = 85) has been recorded across the Malpighian tubules of Locusta. The effect of varying the Na⁺ and K⁺ concentration in the bathing medium on the transepithelial potential has been determined. The data show that the transepithelial potential does not obey the Nernst equation for K⁺. Ouabain, ethacrynic acid and amiloride all inhibit the transepithelial potential. The results are discussed in relation to the nature of the mechanisms of cation transport across the Malpighian tubules.     

Secretory activity of the segmented Malipighian tubules of Cenocorixa bifida (Hung.) (Hemiptera,Corixidae)

In vitro studies with whole Malpighian tubules of the water boatman Cenocorixa bifida show that the composition of the secreted fluid is similar to that reported in several other insects. All three distal segments of the tubule are secretory, but the rate of fluid, sodium, potassium and chloride contribution is greatest in the short, most distal segment (IV). The sodium, potassium, and chloride concentrations of fluid produced by segments II, III, and IV change sequentially in a gentle stepwise gradient; osmotic pressure does not change significantly. Segment II produces fluid with a high pH under cAMP stimulation thus raising the pH of fluid produced by the whole tubule. Segment III is the site of dye transport and the production of luminal secretory granules. Possible correlations between the morphology and transport properties of the different segments are discussed.     

Feulgen-DNA response and chromatin condensation in Malpighian tubules of Melipona rufiventris and Melipona quadrifasciata (Hymenoptera, Apoidea)

Melipona quadrifasciata and Melipona rufiventris are stingless bee species which present low and high heterochromatin content, respectively, on their mitotic chromosomes as assessed visually after a C-banding assay. However, these species do not show differences in the C-banding responses of their Malpighian tubule interphase nuclei. In the present study, the Feulgen-DNA response, which could inform on differences in DNA depurination due to differences in chromatin condensation, was compared in the cell nuclei of the Malpighian tubules of these species. It was hypothesized that differences in acid hydrolysis kinetics patterns, as assessed by Feulgen reaction and studied microspectrophotometrically, could discriminate M. quadrifasciata and M. rufiventris interphase nuclei not distinguishable with the C-banding method. Feulgen-DNA values corresponding to more than one ploidy class were found in both species; these values at the hydrolysis time corresponding to the maximal DNA depurination for each ploidy degree were higher in M. quadrifasciata, reflecting a higher DNA content in the Malpighian tubule cell nuclei of this species compared to those of M. rufiventris at the same larval instar. The maximal Feulgen-DNA values of M. quadrifasciata after short (50 min) and long (90 min) hydrolysis times were found to be closer to each other, while those of M. rufiventris occurred sharply at the long hydrolysis time, indicating that DNA depurination in M. quadrifasciata occurred faster. This result is probably related to the involvement of differences in chromatin condensation; it agrees with the idea that M. rufiventris contains more heterochromatin than M. quadrifasciata, which is supported by the analysis of results obtained with the image analysis parameter average absorption ratio. The depurination kinetics studied here with the Feulgen reaction were revealed to be more pertinent than the C-banding technique in establishing differences in levels of chromatin condensation for these cell nuclei.     

The effects of altered expression of the Keap1/CncC pathway on the secretion of fluid and salicylate by Malpighian tubules of Drosophila melanogaster

The Keap1‐Nrf2 pathway is a major upstream regulator of xenobiotic detoxification. In Drosophila melanogaster Meigen, targeted expression of Keap1 and CncC (the latter the orthologue of human Nrf2) in the Malpighian (renal) tubules is known to confer resistance to lethal doses of the pesticide malathion, which is metabolized into organic anions. Dietary exposure to organic anions such as salicylate (10 mm ) causes increases in the fluid secretion rate and salicylate flux across Malpighian tubules. In the present study, salicylate‐selective microelectrodes and Ramsay assays are used to determine the role of Keap1/Nrf2 in regulating these responses. Genetic manipulations designed to increase Nrf2 activity (by knockdown of the repressor Keap1 or overexpression of the Nrf2 coactivator MafS) or to decrease Nrf2 activity (by overexpression of Keap1) are also studied. Although the results of the Keap1 manipulations are inconclusive, there is no increase in the fluid secretion rate or salicylate flux in tubules isolated from flies in which MafS expression is increased.     

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.     

Ultrastructural studies on the Malpighian tubule of the pill millipede,Glomeris marginata (Villers)

Summary Ultrastructural studies on the Malpighian tubules of Glomeris marginata (Villers) reveal considerable morphological differences between the upper, fluid secreting, segment, and the lower segment which is at present of unknown function. Previous reports have shown that the upper tubule has a high permeability to compounds of high molecular weight. This may be accounted for by the fact that the epithelium shows very extensive intercellular spaces which are linked directly to junctions apparently specialised to provide a low resistance extracellular pathway between the haemocoel and the tubule lumen.Histochemical studies on the localisation of phosphatase enzymes reveal intracellular vesicles with acid phosphatase activity. The basal labyrinth of the lower tubule exhibits considerable alkaline phosphatase activity which is apparently identical in location to the enzyme revealed by two different ATPase localisation techniques.The authors are indebted to the Science Research Council for financial supportThe authors wish to thank Mrs. Margarita Petri for her technical assistance and advice     

Serotonin has kinin-like activity in stimulating secretion by Malpighian tubules of the house cricket Acheta domesticus

Serotonin stimulates secretion by Malpighian tubules (MT) of a number of insects, and functions as a diuretic hormone in Rhodnius prolixus and in larval Aedes aegypti. Serotonin is here shown to be a potent stimulant of secretion by MT of the house cricket, Acheta domesticus, with an apparent EC₅₀ of 9.4 nmol L⁻¹, although its diuretic activity is just 25% of the maximum achievable with either the native CRF-related peptide, Achdo-DH, or a crude extract of the corpora cardiaca. In this respect, the diuretic activity of serotonin is similar to that of the cricket kinin Achdo-KI, and when tested together their actions are not additive, which suggests they target the same transport process. Consistent with this suggestion, the activity of serotonin is chloride-dependent and is associated with a non-selective stimulation of NaCl and KCl transport. In common with Achdo-KI, serotonin has no effect on cAMP production by isolated MT, and both act synergistically with exogenous 8bromo-cAMP in stimulating fluid secretion, most likely by promoting the release of Ca²⁺ from intracellular stores. A number of serotonin agonists and antagonists were tested to determine the pharmacological profile of receptors on cricket MT. The results are consistent with the diuretic activity of serotonin being mediated through a 5-HT₂-like receptor.     

庭疾灶螽中肠及马氏管结构

【目的】本研究旨在以庭疾灶螽Tachycines asynamorus为例探索驼螽消化系统和排泄系统在结构上与其生活环境的适应关系。【方法】运用解剖学方法、石蜡切片技术、冰冻切片技术及超薄切片技术对庭疾灶螽中肠及马氏管的结构进行研究。【结果】庭疾灶螽中肠向前延伸出3个胃盲囊包围着前胃。中肠上皮由再生细胞、柱状上皮细胞和内分泌细胞构成,具有典型的再生细胞龛;闭合型内分泌细胞紧贴在再生细胞龛的外围,基底区聚集大量的分泌颗粒。柱状上皮细胞内聚集有2类大的分泌颗粒：线团状颗粒和电子密度很高的球状颗粒;中肠管腔内有明显的围食膜结构,中肠基底部由基膜和肌肉层组成。马氏管着生在中后肠的交界处,从横切面看马氏管管壁具有3~5个细胞,细胞近管腔端部具有大量长微绒毛,细胞质内分布着电子致密的同心圆球晶体,基底膜内折形成膜迷路。【结论】庭疾灶螽中肠柱状上皮细胞的线团状颗粒由微丝包裹;内分泌细胞由再生细胞龛中的细胞分化而来,产生内分泌颗粒并将其排到血腔;中肠基膜发达,包含微丝与复合糖成分,基膜通过对中肠上皮细胞的支撑作用为肠道蠕动提供保障。庭疾灶螽马氏管细胞中可见大量颗粒和大量同心圆球晶体,推测可能是一种储存排泄。     

Stimulated fluid secretion is sodium dependent in the Malpighian tubules of Locusta migratoria

The ionic dependencies of stimulated and unstimulated Locusta tubules have been studied. K⁺, Na⁺, Cl^? are essential to both basal and stimulated secretion. K⁺ is secreted against a concentration gradient in unstimulated tubules. In response to diuretic hormone or cAMP application, there is a dramatic influx of K⁺ into the lumen. A high level of Na⁺ and Cl^? in the bathing medium is required to allow maximal fluid secretion. The tubules show an apparent impermeability to Na⁺; its concentration in the secreted fluid is always much less than in the bathing medium. If Na⁺ is omitted from the medium and excess K⁺ added (80 mM K), then although basal secretion occurs (2.5 nl/min), the tubules fail to respond to stimulation. Clearly Na⁺ has an important indirect role to play in stimulated fluid secretion.     

The presence of N-acetylneuraminic acid in Malpighian tubules of larvae of the cicada Philaenus spumarius

Sialic acid-containing glycoconjugates are generally considered to be unique to the deuterostomes, a lineage of the animal kingdom which includes animals from the echinoderms up to the vertebrates. There are, however, two isolated reports of sialic acid occurring in the insect species Drosophila melanogaster and Galleria mellonella. Since insects are classified as protostomes, these findings call previous assumption on the phylogenetic distribution and thus on the evolution of sialic acids into question. Here, we report the occurrence of N-acetylneuraminic acid (Neu5Ac) in larvae of the cicada Philaenus spumarius. Cytochemical analysis of larval sections with lectins from Sambucus nigra and Limax flavus suggested the presence of sialic acids in the concrement vacuoles of the Malpighian tubules. The monoclonal antibody MAb 735, which is specific for polysialic acid, labelled the same structures. A chemical analysis performed by HPLC of fluorescent derivatives of sialic acids and by GLC-MS provided sound evidence for the presence of Neu5Ac in the Philaenus spumarius larvae. These data suggest that in this cicada Neu5Ac occurs in 2,8-linked polysialic acid structures and in 2,6-linkages. The results provide further evidence for the existence of sialic acids in insects and in linkages known to occur in glycoconjugates of deuterostomate origin.     

Ultrastructural tracer studies on the permeability of the Malpighian tubule of the Pill Millipede,Glomeris marginata (Villers)

Summary The electron-dense tracers ferritin, and iron-dextran, and the protein horseradish peroxidase, have been used to investigate the ultrastructural basis of permeability in the upper and lower segments of the Malpighian tubules of Glomeris marginata. All these materials were able to cross the basal lamina and enter the tubule lumen of the upper segment, and it was established that horseradish peroxidase was able to enter the channels which interrupt the apical junctions.In the upper segment, ferritin, iron-dextran, and horseradish peroxidase are all taken up and accumulated within intracellular vesicles. In the lower segment ferritin and iron-dextran enter the cells but become generally distributed over the cyptoplasm, as well as entering membrane-bounded vacuoles. The behaviour of horseradish peroxidase could not be assessed owing to the presence of endogenous peroxidase activity in the cells.After fixation by direct application of glutaraldehyde to the undissected tubules, the extracellular spaces contained large numbers of membrane-bounded vesicles. The significance of these observations is discussed in relation to the physiological activities of the tubules.The authors are indebted to the Science Research Council for financial supportThe authors wish to thank Mrs. Margarita Petri for her technical assistance and advice     

The paracellular channel for water secretion in the upper segment of the Malpighian Tubule of Rhodnius prolixus

Lumen to bath J ₁₂/C ₁ and bath to lumen J ₂₁/ C ₂ fluxes per unit concentration of 19 probes with diameters (d ^m) ranging from 3.0–30.0 Å (water, urea, erythritol, mannitol, sucrose, raffinose and 13 dextrans with d ^m 9.1–30.0 Å) were measured during volume secretion (J _v ) in the upper segment of the Malpighian Tubule of Rhodnius by perfusing lumen and bath with ¹⁴C or ³H-labeled probes. J _net=(J ₁₂/C ₁ – J ₂₁/C ₂) was studied as a function of J _v · J _v was varied by using different concentrations of 5-hydroxy tryptamine. J _net for ³H-water was not different from J _v We found: (i) A strong correlation between J _net and J _v for 8 probes d ^m =3.0–11.8 Å (group a probes), indicating that the convective component of J _net is more important than its diffusive component and than unstirred layers effects which are negligible. Therefore group a probes are solvent dragged as they cross the epithelium, (ii) There is no correlation between J _net and J _v for 11 probes with d ^m=11.8–30 Å (group b). Therefore these probes must cross the epithelium by diffusion and not by solvent drag, (iii) In a plot of J _net/J _v vs. d ^m group a probes show a steep linear relation with a slope = –0.111, while for group b probes the slope is –0.002. Thus there is a break between groups a and b in this plot. We tried to fit the data with models for restricted diffusion and convention through cylindrical or parallel slit pathways. We conclude that (i) group a probes are dragged by water through an 11.0 Å-wide slit, (ii) Most of J _v must follow an extracellular noncytosolic pathway, (iii) Group b probes must diffuse through a 42 Å-wide slit, (iv) A cylindrical pathway does not fit the data.E.G. is a Visiting Scientist at IVIC. It is a pleasure to thank Drs. A.E. Hill and Bruria Shachar-Hill for their suggestion of the use of dextrans, their instruction and help with the dextran separation technique, and their extensive discussions. Dr. R. Apitz, Mr H. Rojas and Mrs. Fulvia Bartoli were most helpful with suggestions during the course of the experimental work. Mr. Jose Mora was fundamental help with the equipment. Mrs. Lelis Ochoa and Mr. Luis F. Alvarez helped with some of the drawings. This work was partially supported by CONICIT, Fundación Polar and CDCH of UCV. It is a pleasure to thank Dr. H. Passow and Dr. K.J. Ullrich at the Max Planck Institut für Biophysik (Frankfurt/Main) where this work was initiated.