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     

Evidence for hormonal control of diuresis after a blood meal in the mosquito Aedes aegypti

In previous studies we have presented evidence for the role of peptides, isolated from heads of the mosquito Aedes aegypti, in stimulating fluid secretion by isolated Malpighian tubules. In the present study we conducted experiments to investigate whether these peptides are involved in hormone-mediated diuresis after a blood meal. In vivo experiments showed that the head was required to maintain diuresis after the blood meal. Whereas feeding on blood triggered a prompt diuresis in the intact mosquito, subsequent decapitation caused a gradual, not an abrupt, decline in urine excretion rate. Hemolymph collected from mosquitoes fed blood significantly stimulated fluid secretion in vitro by isolated Malpighian tubules, whereas hemolymph from unfed or blood-fed decapitated mosquitoes did not. These results indicate that a diuretic factor was released into the hemolymph after a blood meal. This factor was not present in the hemolymph of decapitated females. We identified the head as a source of diuretic factors. Peptides isolated from a head extract by high-performance liquid chromatography, when injected into the hemocoel of blood-fed decapitated mosquitoes, triggered diuresis in vivo and also stimulated fluid secretion in isolated Malpighian tubules. These studies support the hypothesis that the head is a storage site for diuretic peptides that may be released after a blood meal to control diuresis.     

Effects of lignoids on a hematophagous bug, Rhodnius prolixus: feeding, ecdysis and diuresis

The effects of lignoids on feeding, ecdysis and diuresis in fourth-instar larvae of Rhodnius prolixus (Hemiptera) were investigated. Up to 100 microg/ml burchellin, podophyllotoxin, pinoresinol, sesamin, licarin A, or nordihydroguaiaretic acid (NDGA) in the diet did not induce antifeedant effects. Pinoresinol and NDGA significantly inhibited ecdysis. In experiments in vivo, burchellin and podophyllotoxin reduced the production of urine after feeding. 5-Hydroxytryptamine (5-HT), a diuretic hormone, partially counteracted this effect of burchellin. In experiments in vitro, using isolated Malpighian tubules, (i) burchellin reduced diuretic hormone levels in the hemolymph but not the amount of diuretic hormone stored in the thoracic ganglionic masses (including axons), (ii) burchellin decreased the volume of urine secreted by isolated Malpighian tubules, and (iii) 5-HT could not overcome the effect of burchellin upon the Malpighian tubules. We conclude that burchellin interfered with the release, but not with the production of diuretic hormone by the thoracic ganglionic mass or induced an antidiuretic hormone and directly affected the Malpighian tubules.     

5-hydroxytryptamine regulates the (Na++K+)ATPase activity in malpighian tubules of Rhodnius prolixus: Evidence for involvement of G-protein and cAMP-dependent protein kinase

5-Hydroxytryptamine (5-HT, serotonin) acts as a diuretic hormone in Rhodnius prolixus, where it increases to 0.1 μM in the haemolymph during feeding and stimulates the fluid secretion in isolated Malpighian tubules. The ouabain-sensitive (Na⁺+K⁺)ATPase activity present in homogenates of Malpighian tubules from unfed Rhodnius prolixus is inhibited 60% by 0.01 μM 5-HT. This inhibition is reversed by ketanserin, a 5-HT₂ receptor antagonist in mammals, and also by GDPβS, a competitive inhibitor of G-protein GTPase activity. GTPγS, a nonhydrolysable analog of GTP, and cholera toxin, a G_s-protein activator, also inhibit the ouabain-sensitive (Na⁺+K⁺)ATPase activity, while pertussis toxin, a G_i-protein inhibitor, has no effect. The (Na⁺+K⁺)ATPase activity is inhibited 55% by 0.4–100 μM dibutyryl-cAMP in the presence of IBMX, a phosphodiesterase inhibitor, which also potentiates the effect of a low concentration of 5-HT. The cAMP-dependent protein kinase inhibitor peptide abolishes the 5-HT effect. These data suggest that the (Na⁺+K⁺)ATPase activity in Malpighian tubules is inhibited by 5-HT through activation of G_s-protein and a cAMP-dependent protein kinase. Inhibition of the Na⁺+K⁺ pump would contribute to the diuretic effect of 5-HT. Arch. Insect Biochem. Physiol. 36:203–214, 1997. © 1997 Wiley- Liss, Inc.     

5-Hydroxytryptamine stimulates fluid secretion in locust malpighian tubules independently of cAMP

5-Hydroxytryptamine (5-HT) stimulates fluid secretion by semi-isolated Malpighian tubules of Locusta in a dose-dependent manner. The threshold of stimulation is between 10(-8) and 10(-7) M 5-HT; maximal activation occurs at doses greater than 10(-6) M. Relative to the activation induced by diuretic hormone (storage lobe extracts), 5-HT increases the rate of fluid secretion by only 65%. Phentolamine, the alpha-adrenergic blocker, failed to inhibit either DH or 5-HT stimulated secretion. Diuretic hormone raises the levels of intracellular of cAMP, and activates adenylate cyclase in plasma membrane preparations of Locusta Malpighian tubules. 5-HT (10(-4) M) has no effect in either assay system. Thus 5-HT can stimulate fluid secretion independently of cAMP. A hypothetical model for hormone stimulated fluid secretion by Locusta Malpighian tubules, involving dual-receptor activation, is proposed. Other biogenic amines, including octopamine, adrenalin, dopamine, synephrine and the formamidine chlordimeform were tested for their ability to stimulate fluid secretion. Only dopamine showed a weakly stimulatory effect.     

Investigations of the signaling cascade involved in diuretic hormone stimulation of Malpighian tubule fluid secretion in Rhodnius prolixus

In insects, the excretory system is comprised of the Malpighian tubules (MTs) and the hindgut, which collectively function to maintain ionic and osmotic balance of the haemolymph and rid the organism of toxic compounds or elements in excess. Secretion by the Malpighian tubules of insects is regulated by a variety of hormones including peptidergic factors as well as biogenic amines. In Rhodnius prolixus, two endogenous diuretic hormones have been identified; the biogenic amine serotonin (5-hydroxytryptamine, 5-HT) and the corticotropin releasing factor-related peptide, RhoprCRF. Both factors significantly increase secretion by MTs and are known to elevate intracellular levels of cAMP. Interestingly, applying sub-maximal doses of these two diuretic factors in combination on isolated MTs in vitro reveals synergistic effects as rates of fluid secretion are significantly higher than would be expected if rates of secretion from MTs treated with each factor alone were summed. This observed synergism suggests that different downstream targets may be activated by the two diuretic factors, but that some cellular elicitors may be shared since cAMP is elevated in response to either diuretic hormone.     

Diuretic activity of Mas-DP II,an identified neuropeptide from Manduca sexta: An in vivo and in vitro examination in the adult moth

Mas-DP II, a recently identified 30 amino acid diuretic peptide isolated from the tobacco hornworm moth, Manduca sexta, was tested for its ability to increase fluid excretion in adult M. sexta, and for the ability to elevate the rate of fluid secretion from isolated Malpighian tubules cultured in vitro. Mas-DP II was found to increase fluid weight loss from decapitated adult moths in a dose-dependent manner; weight loss increased significantly at doses as low as 5 ng for female moths and 25 ng for male moths. Male moths injected with large doses of Mas-DP II continued to exhibit increased rates of fluid loss up to 4 h post-injection. In vitro, Mas-DP II stimulated fluid secretion from isolated Malpighian tubules at concentrations as low as 4 nM for tubules from both male and female moths. © 1994 Wiley-Liss, Inc.     

Secretion of water and ions by malpighian tubules of larval mosquitoes: effects of diuretic factors, second messengers, and salinity

The effects of changes in the salinity of the rearing medium on Malpighian tubule fluid secretion and ion transport were examined in larvae of the freshwater mosquito Aedes aegypti and the saltwater species Ochlerotatus taeniorhynchus. For unstimulated tubules of both species, the K(+) concentration of secreted fluid was significantly lower when larvae were reared in 30% or 100% seawater (O. taeniorhynchus only), relative to tubules from freshwater-reared larvae. The Na(+) concentration of secreted fluid from unstimulated tubules of O. taeniorhynchus reared in 30% or 100% seawater was higher relative to tubules from freshwater-reared larvae. The results suggest that changes in salinity of the larval rearing medium lead to sustained changes in ion transport mechanisms in unstimulated tubules. Furthermore, alterations of K(+) transport may be utilized to either conserve Na(+) under freshwater (Na(+)-deprived) conditions or eliminate more Na(+) in saline (Na(+)-rich) conditions. The secretagogues cyclic AMP [cAMP], cyclic GMP [cGMP], leucokinin-VIII, and thapsigargin stimulated fluid secretion by tubules of both species. Cyclic AMP increased K(+) concentration and decreased Na(+) concentration in the fluid secreted by tubules isolated from O. taeniorhynchus larvae reared in 100% seawater. Interactions between rearing salinity and cGMP actions were similar to those for cAMP. Leucokinin-VIII and thapsigargin had no effect on secreted fluid Na(+) or K(+) concentrations. Results indicate that changes in rearing medium salinity affect the nature and extent of stimulation of fluid and ion secretion by secretagogues.     

The distribution of a kinin-like peptide and its co-localization with a CRF-like peptide in the blood-feeding bug, Rhodnius prolixus

Rhodnius prolixus, a blood-feeding hemipteran insect, ingests large meals which are followed by rapid diuresis to eliminate excess water and salt. In Rhodnius, serotonin and an unidentified peptide(s) [33,34] have been shown to act as neurohormonal diuretic factors. In other insects, two families of diuretic peptides, the corticotropin releasing factor (CRF)-like, and kinin peptides [9], have been identified and sequenced. Recently, we demonstrated the presence of a CRF-like diuretic peptide in the CNS and digestive system of Rhodnius [47] using immunohistochemistry and bioassay.In this study, combining immunohistochemistry and radioimmunoassay (RIA) techniques, we show the presence of leucokinin-like peptide(s) in the CNS and digestive system of Rhodnius 5th instar. Additionally, double-label immunohistochemistry demonstrates that the leucokinin-like and CRF-like peptides are co-localized in the posterior lateral neurosecretory cells of the mesothoracic ganglionic mass (MTGM) and in neurohaemal areas on abdominal nerves one and two, suggesting the possibility of co-release of the peptides into the hemolymph.Partially purified extracts of the CNS and neurohaemal tissue were tested in vitro on Malpighian tubule secretion and cAMP assays. The factors eluting with increasing acetonitrile percentages from Sep-Pak cartridges were assayed in the presence or absence of ketanserin, a serotonin antagonist which blocks the effects of serotonin on Malpighian tubules. The results indicate activity of serotonin and a CRF-like diuretic peptide on Rhodnius Malpighian tubules, but fail to demonstrate activity of the leucokinin-like peptide(s).The rapid diuresis following feeding is a highly coordinated event, requiring the movement of water and salt across the epithelial cells of the crop into the hemolymph, and from the hemolymph across the cells of the Malpighian tubules. The urine then travels along the Malpighian tubules into the hindgut in order to be expelled. The presence of a leucokinin-like peptide(s) in the CNS and digestive system, which co-localizes with a CRF-like peptide(s), suggests that kinins may play a role in the rapid diuresis, although possibly not directly on the Malpighian tubules.     

The physiological response of larval <Emphasis Type="Italic">Chironomus riparius</Emphasis> (Meigen) to abrupt brackish water exposure

The physiological response of larval Chironomus riparius was examined following direct transfer from freshwater (FW) to brackish water (BW; 20% seawater). Endpoints of hydromineral status (hemolymph Na⁺, Cl⁻, and K⁺ levels, hemolymph pH, body water content, and whole body Na⁺/K⁺-ATPase and V-type H⁺-ATPase activity) were examined 1, 3, 5, 12 and 24 h following BW transfer. Larvae transferred from FW to FW served as a control. Hemolymph Na⁺ and Cl⁻ levels increased following BW transfer. Hemolymph pH was initially regulated, but significantly decreased after 24 h in BW. Changes in hemolymph ions were not caused by osmotic loss of water from the hemolymph, since larvae tightly regulated total body moisture content. Furthermore, salinity did not affect hemolymph K⁺. When larvae were transferred to BW, Na⁺/K⁺-ATPase (NKA) activity did not significantly alter relative to FW control animals. In contrast, V-type H⁺-ATPase (VA) activity in C. riparius significantly decreased in BW. In FW-reared C. riparius, whole body NKA and VA activities were equivalent. However, in the isolated gut with intact Malpighian tubules of FW-reared C. riparius, VA activity was significantly greater than whole body while NKA activity was equivalent. This suggested that gut and/or Malpighian tubule VA activity contributes significantly to whole body VA activity and that a decline in whole body VA activity in BW may be closely linked to alterations in the physiology of gut and Malpighian tubule tissue. Taken together, data indicate that VA is important for ion uptake in FW and that the NKA does not play a major role in regulating ion homeostasis when larvae are acutely exposed to BW.     

Mode of action studies on a Manduca sexta diuretic hormone

The mode of action of a diuretic hormone from pharate adult Manduca Sexta heads, which triggers fluid loss in M. sexta larvae and Pieris rapae adults, was studied. In vivo, Mas-DH (M. sexta diuretic hormone) decreased fluid absorption from larval recta, and increased levels of the second messenger cAMP in recta and Malpighian tubules (Mt) from larvae, and in fat body of larvae and adult M. sexta. In vitro, Mas-DH triggered minor changes in fluid loss from adult Mt, but did not affect levels of cAMP in Mt from larvae, pharate adults, or adults, though it elevated cAMP levels in fat body of these stages. © 1992 Wiley-Liss, Inc.     

Effects of acute or chronic exposure to dietary organic anions on secretion of methotrexate and salicylate by Malpighian tubules of Drosophila melanogaster larvae

The effects of dietary exposure to organic anions on the physiology of isolated Malpighian tubules and on tubule gene expression were examined using larvae of Drosophila melanogaster. Acute (24 h) or chronic (7 d) exposure to type I organic anions (fluorescein or salicylate) was associated with increased fluid secretion rates and increased fluxes of both salicylate and the type II organic anion methotrexate. By contrast, chronic exposure to dietary methotrexate was associated with increased fluid secretion rate and increased flux of methotrexate, but not salicylate. Exposure to methotrexate in the diet resulted in increases in the expression of a multidrug efflux transporter gene (MET; CG30344) in the Malpighian tubules. There were also increases in expression of genes for either a Drosophila multidrug resistance–associated protein (dMRP; CG6214) or an organic anion transporting polypeptide (OATP; CG3380), depending on the concentration of methotrexate in the diet. Exposure to salicylate in the diet was associated with an increase in expression of dMRP and with decreases of MET and OATP. Exposure to dietary salicylate or methotrexate was also associated with different patterns of expression of heat shock protein genes. The results suggest that exposure to specific type I or type II organic anions has multiple effects and results not only in increased organic anion transport but also in increased rates of inorganic ion transport, which drives osmotically‐obliged fluid secretion. Increased fluid secretion may enhance secretion of organic anions by eliminating diffusive backflux from the tubule lumen to the hemolymph. © 2010 Wiley Periodicals, Inc.     

The influence of canavanine and related compounds on the water balance system of locusts

In vivo increase in haemolymph volume of canavanine-treated locusts substantiates our previous in vitro findings that canavanine inhibits fluid secretion by locust Malpighian tubules. Furthermore when diuretic hormone is applied in vivo after canavanine treatment haemolymph volume is drastically reduced below levels retained in locusts untreated with canavanine. Again this is in accord with canavanine potentiation of semi-isolated Malpighian tubules and enhanced fluid secretion in vitro. The response is specific to canavanine; compounds similar in structure (arginine, argininic acid, citrulline, canaline, ornithine and homoserine) have no effect on the rate of fluid secreted by Malpighian tubules. Only partial competition is obtained with uridine homoserine.     

Effects of dietary or injected organic cations on larval Drosophila melanogaster: mortality and elimination of tetraethylammonium from the hemolymph

This study of larval Drosophila melanogaster examined the effects of injecting the prototypical organic cation tetraethylammonium (TEA) into the hemocoel or adding TEA and/or other organic cations to the diet. Mortality, hemolymph TEA levels, and Malpighian tubule TEA secretion rates were measured. The LD50 for dietary TEA was 158.4 mM and mortality increased if competitive inhibitors of organic cation transporters were also included in the diet. Mortality increased from 24% on TEA (100 mM) alone to 83 and 67% when the diet contained both TEA and quinidine (10 mM) or cimetidine (100 mM), respectively. TEA-selective microelectrode measurements indicated that hemolymph TEA concentration was approximately 3% of that in the diet for larvae maintained on TEA-enriched diet for 24 h. Malpighian tubules isolated from larvae exposed to dietary TEA excreted more TEA than did tubules from controls fed a TEA-free diet. However, the rate of decline of hemolymph TEA concentration following ingestion or injection of TEA into the hemocoel was greater than that explicable by rates of active transport by the gut and Malpighian tubules (MTs). We propose that TEA concentrations in the hemolymph are reduced not only by active transport across the MTs and gut, but also by diffusion into the gut. The latter pathway is particularly important when larvae previously maintained upon TEA-enriched diet are transferred to a TEA-free diet. The ingestion of TEA-free food not only clears the gut lumen, but also creates a TEA-free compartment into which TEA may passively diffuse from the hemolymph.     

Developmental changes in Malpighian tubule fluid transport

Developmental changes in Malpighian tubule fluid transport were studied using in vitro and in situ preparations from timed larval, pupal and adult skipper butterflies (Calpodes ethlius). The ability to transport fluid and the rate of fluid secretion depend on the developmental stage and the physiological state. Larval tubules are permanently switched on, do not require a diuretic hormone and transport fluid at an increasing rate as the larvae feed and grow. Fluid transport continues at larval-larval moults but is switched off 24 hr before pupal ecdysis. No secretory activity occurs during the first half of the pupal stage when the tubules are remodelled for adult function. The resumption of fluid transport midway through the pupal stage is in preparation for a rapid diuresis at adult emergence. High rates of fluid secretion are associated with feeding or drinking in adults.     

Body extracts from the forest ant and honeybee: chromatographic comparison of factors affecting the ant Malpighian tubule

Abstract.  Crude trifluoroacetic acid extracts were prepared from different body parts of the forest ant, Formica polyctena Foerster, Hymenoptera, Formicidae, and the honeybee, Apis mellifera carnica Pollmann, Hymenoptera, Apidae. Extracts were prepurified by means of solid phase extraction over reversed-phase cartridges. The effects of the resulting fractions on fluid secretion rates were tested on single preparations of ant Malpighian tubules. Diuretic and antidiuretic factors were shown to be present. High-performance liquid chromatography allowed for the identification of a diuretic factor from the bee-head extract which had a similar elution profile to a previously purified diuretic peptide from ant-head extracts. An antidiuretic factor was also identified from the bee abdominal extract. Similar to an antidiuretic peptide, which was purified from ant abdominal extracts, this antidiuretic factor inhibited fluid secretion and depolarized the transepithelial potential of ant Malpighian tubules.     

Beetle diuretic peptides: the response of mealworm (Tenebrio molitor) Malpighian tubules to synthetic peptides, and cross-reactivity studies with a dung beetle (Onthophagus gazella)

This paper reports the effects of different diuretic factors on the Malpighian tubules of beetles. Calcitonin (CT)-like peptides from silkmoth and mosquito increase fluid secretion in a dose-dependent manner in the tubules of Tenebrio molitor, but the cockroach CT-like peptide, Dippu-DH(31), has no effect. Thapsigargin induces a small but significant increase in tubule secretion rates. The interactions between different factors in mealworm tubules were explored by testing CT-like peptides, thapsigargin and the mealworm CRF-related diuretic factor Tenmo-DH(37) in various combinations, but no synergistic effects were observed. C-terminal fragments of the CRF-related diuretic peptides Locmi-DH(46) and Dippu-DH(46) fail to increase fluid secretion in mealworm tubules, unlike their corresponding whole peptides. Cross-reactivity of factors between beetle species was investigated using the scarabaeid Onthophagus gazella. Tenmo-DH(37) increases fluid secretion in isolated tubules of O. gazella in a dose-dependent manner, revealing a high degree of cross-reactivity in this distantly related beetle species. However, homogenates of O. gazella brains inhibited fluid secretion in mealworm tubules.     

Diuresis or clearance: Is there a physiological role for the “diuretic hormone” of the desert beetle Onymacris?

Malpighian tubules of Namib Desert tenebrionid beetles of the genus Onymacris are strongly stimulated by homogenates of the corpora cardiaca. The corpora cardiaca of other arid-adapted tenebrionids also contain diuretic material. Biogenic amines, which could be released during the preparation of corpora cardiaca extracts, do not stimulate fluid secretion in tubules of Onymacris rugatipennis. The diuretic factor in corpora cardiaca extracts is stable to boiling and to incubation with pronase. HPLC separation of the corpora cardiaca of O. rugatipennis gives a single region with diuretic activity in both secretory and electrical bioassays. Diuretic activity can not be detected in the haemolymph of Onymacris, and injection of corpora cardiaca extracts into the beetles does not cause diuresis. Simultaneous injection of corpora cardiaca and the dye amaranth shows that the most of the dye transported by the Malpighian tubules moves anteriorly into the midgut, indicating fluid recycling by this route. The most likely function for this “diuretic hormone” is clearance of metabolic wastes from the haemolymph.     

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.