Transepithelial transport of salicylate by the Malpighian tubules of insects from different orders

The organic anion salicylate is a plant secondary metabolite that protects plants against phytophagous insects. In this study, a combination of salicylate-selective microelectrodes and a radioisotope tracer technique was used to study the transepithelial transport of salicylate by the Malpighian tubules of 10 species of insects from five orders. Our results show that salicylate is transported into the lumen of the Malpighian tubules in all the species evaluated, except Rhodnius prolixus. The transepithelial transport of salicylate by the Malpighian tubules of Drosophila simulans, Drosophila erecta, Drosophila sechellia, and Acheta domesticus was saturable, Na⁺-dependent and inhibited by α-cyano-4-hydroxycinnamic acid. This transport system resembles that previously found in tubules of Drosophila melanogaster. In contrast, transepithelial transport of salicylate by Malpighian tubules of Tenebrio molitor, Plagiodera versicolora, Aedes aegypti, and Trichoplusia ni was unaffected by Na⁺-free bathing saline. The presence of both salicylate and salicylate metabolites in the secreted fluid samples from the Malpighian tubules of A. domesticus, R. prolixus, T. molitor, and T. ni indicates that insect Malpighian tubules may both transport and metabolize salicylate. The highest capacities to rid the hemolymph of salicylate were found in T. molitor, P. versicolora and Drosphila spp. Our results suggest that transport of salicylate by the Malpighian tubules might contribute to elimination of this organic anion from the hemolymph, particularly in some species that encounter high levels of organic anion in the diet.     

Water and solute transport by the Malpighian tubules of the stick insect,Carausius morosus

Summary Structural features of the principal, urine-secreting cells (type 1 cells) of the Malpighian tubules of Carausius are de scribedquantitatively and discussed in relation to possible mechanisms of water and solute transport. Mitochondria are arranged in two bands of about equal volume near to the basal and apical surfaces, suggesting active processes occur at both surfaces. Basal infoldings and apical microvilli which greatly amplify the cell surface are probably primarily devices to increase the passive permeability of the tissue to solutes. They do not provide functionally significant standing-osmotic-gradients. The extensive endoplasmic reticulum is locally differentiated into several components and ramifies between the infoldings and along microvilli but probably is not an intracellular conduit for the majority of urinary constituents. Vesicles and stages in their formation or liberation are observed both basally and apically although they probably do not contribute significantly to transcellular transport. At present it remains a problem to satisfactorily account for observations that the urine of Carausius can be hypotonic.This investigation formed part of a dissertation for the degree of Ph. D. in the University of Newcastle upon Tyne. It is a pleasure to thank Prof. J. Shaw for his advice and encouragement and the Science Research Council for financial support.     

The fine structure of the type 2 cells in the Malpighian tubules of the stick insect,Carausius morosus

Summary Type 2 cells, or mucocytes, are present in both inferior and superior Malpighian tubules ofCarausius morosus and are concentrated towards the distal ends of the main urine-secreting parts. They are absent from the proximal few millimetres of the main part and from the distal specialised regions of the tubules. They possess numerous Golgi bodies and abundant granular E. R., which is consistent with the hypothesis that they secrete mucus. However, they possess basal infoldings and apical microvilli suggesting that they may transport substances across the tubule wall. It is suggested that they perform both functions. Reabsorption of ions or water could precipitate solid components of the urine (e.g., uric acid). Mucus may be important in nucleation of crystalline material and also prevent abrasion of the brush border.This investigation formed part of a thesis for the degree of Ph.D. in the University of Newcastle upon Tyne. It is a pleasure to thank Professor J. Shaw for his advice and encouragement and the Science Research Council for financial support.     

Calcium transport across the basolateral membrane of isolated Malpighian tubules: a survey of several insect orders

The Malpighian tubules play a major role in haemolymph calcium homeostasis in insects by sequestering excess Ca²⁺ within the biomineralized granules that often accumulate in the tubule cells and/or lumen. Using the scanning ion‐selective microelectrode technique, measurements of basolateral Ca²⁺ transport are determined at several sites along the length of the Malpighian tubules isolated from the eight insects representing seven orders: Drosophila melanogaster (Diptera), Aedes aegypti (Diptera), Tenebrio molitor (Coleoptera), Acheta domesticus (Orthoptera), Trichoplusia ni (Lepidoptera), Periplaneta americana (Blattodea), Halyomorpha halys (Hemiptera) and Pogonomyrmex occidentalis (Hymenoptera). Ca²⁺ transport is specific to tubule segments containing Ca‐rich granules in D. melanogaster and A. aegypti, whereas Ca²⁺ transport is relatively uniform along the length of whole tubules in the remaining species. Generally, manipulation of second messenger pathways using cAMP and thapsigargin has little effect on rates of basolateral Ca²⁺ transport, suggesting that previous effects observed across midtubules of A. domesticus are unique to this species. In addition, the present study is the first to provide measurements of basolateral Ca²⁺ across single principal and secondary tubule cells, where Ca²⁺ uptake occurs only across principal cells. Estimated times for all tubules to eliminate the entire haemolymph Ca²⁺ content in each insect range from 6 min (D. melanogaster) to 19 h (H. halys) or more, indicating that rates of Ca²⁺ uptake by the Malpighian tubules are not always rapid. The results of the present study suggest that the principal cells of the Malpighian tubules contribute to haemolymph calcium homeostasis by sequestering excess Ca²⁺, often within specific tubule segments.     

A new technique for the isolation and purification of the basal lamina from insect tissues

A new technique for the isolation and purification of basal lamina from insect tissues using cell dissociation at pH 2 is described. Tissue incubation in these solutions results in the spontaneous detachment of cells from the basal lamina which can be collected free of any significant contamination by cellular components. Short lengths of plasma membrane which remain attached to the basal lamina can be removed by subsequent sonication or detergent treatment. Using Malpighian tubules as the primary test tissue, we have found that the procedure only requires a few minutes, works equally well on pooled tissue samples, individual tissue pieces or tissue subregions and involves no loss of basal lamina from the starting material.     

Rab11 is required for tubulogenesis of Malpighian tubules in Drosophila melanogaster

Intracellular vesicular trafficking is one of the important tools in maintaining polarity, adhesion, and shape of epithelial cells. Rab11, a subfamily of the Ypt/Rab gene family of ubiquitously expressed GTPases and a molecular marker of recycling endosomes, transports different components of plasma membrane. Here, we report that Rab11 affects tubulogenesis of Malpighian tubules (MTs). MTs are simple polarized epithelial tubular structures, considered as functional analogue of human kidney. Rab11 has pleiotropic effects on MTs development as down‐regulation of Rab11 in principal cells (PCs) of MTs from embryonic stages of development results in reduced endoreplication, clustering of cells, disorganized cytoskeleton, and disruption of polarity leading to shortening of MTs in third instar larvae. Rab11 is also required for proper localization of different transporters in PCs, essential for physiological activity of MTs. Collectively, our data suggest that Rab11 plays a key role in the process of tubulogenesis of MTs in Drosophila.     

Physiological and molecular characterization of methotrexate transport by Malpighian tubules of adult Drosophila melanogaster

A radioisotope tracer technique and quantitative PCR were used to study the mechanisms and regulation of transepithelial transport of the type II organic anion methotrexate (MTX) by the Malpighian tubules of Drosophila melanogaster. Transport of MTX was saturable and Na⁺-independent; the kinetic parameters J_max and K_t were 437 fmol min⁻¹ and 23.5 μM, respectively. The transport of MTX was competitively inhibited by phenol red and probenecid; non-competitively inhibited by salicylate, verapamil and MK-571; and uncompetitively inhibited by Texas Red. Dietary exposure to 0.1 mM MTX led to dramatic increases in gene expression for several members of the ABC family of transporters in both the Malpighian tubules and the gut. Our results suggest that multiple transporters are upregulated in response to dietary exposure to MTX. Increased levels of the protein products which may result from expression of these genes may enhance elimination of toxic compounds such as MTX or its metabolites.     

Mechanisms of calcium sequestration by isolated Malpighian tubules of the house cricket Acheta domesticus

Hemolymph calcium homeostasis in insects is achieved by the Malpighian tubules, primarily by sequestering excess Ca²⁺ within internal calcium stores (Ca‐rich granules) most often located within type I (principal) tubule cells. Using both the scanning ion‐selective electrode technique and the Ramsay secretion assay this study provides the first measurements of basolateral and transepithelial Ca²⁺ fluxes across the Malpighian tubules of an Orthopteran insect, the house cricket Acheta domesticus. Ca²⁺ transport was specific to midtubule segments, where 97% of the Ca²⁺ entering the tubule is sequestered within intracellular calcium stores and the remaining 3% is secreted into the lumen. Antagonists of voltage‐gated (L‐type) calcium channels decreased Ca²⁺ influx ≥fivefold in adenosine 3′,5′‐cyclic monophosphate (cAMP)‐stimulated tubules, suggesting basolateral Ca²⁺ influx is facilitated by voltage‐gated Ca²⁺ channels. Increasing fluid secretion through manipulation of intracellular levels of cAMP or Ca²⁺ had opposite effects on tubule Ca²⁺ transport. The adenylyl cyclase‐cAMP‐PKA pathway promotes Ca²⁺ sequestration whereas both 5‐hydroxytryptamine and thapsigargin inhibited sequestration. Our results suggest that the midtubules of Acheta domesticus are dynamic calcium stores, which maintain hemolymph calcium concentration by manipulating rates of Ca²⁺ sequestration through stimulatory (cAMP) and inhibitory (Ca²⁺) regulatory pathways.     

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.     

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.     

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.     

Ultrastructure of the Malpighian Tubule Cells in the Mosquito Larvae, Anopheles sinesis

Ultrastructure of epithelial cells constituting the Malpighian tubule of Anopheles sinesis last instar larvae was observed with electron microscope. Malpighian tubule consists of four long and narrow tubule structures with principal cells in typical absorptive cells and regenerative cells forming the simple epithelium. Apical plasma membrane of the principal cell is differentiated into microvilli with one mitochondrion in each microvilli. Basal plasma membrane had extreme infolding to form a canaliculi and a well developed mitochondria was attached in the infoldings. And, rER, ribosomes, and vacuoles were well developed inside the cells. However, there were two main cell types depending on the differentiation of cell organelles. Type 1 cell was cubic, forming the distal portion of Malpighian tubule. The length of microvilli was approximately 4 μm and the basal infoldings were introjected to the depth of 2 μm inside the cell. On the other hand, Type II cell that formed the main proxinal portion was a low squamous type cells with shorter 2 μm of microvilli and the basal infoldings were introjected to the depths of 4 μm inside the cell. As for vacuoles scattered inside the cells, they were regularly observed in both Type I and II and the Type II cells had better developed cellular organelles. Although regenerative cells were extremely small, their cellular organelles were developed and their overall electron density was high that they appeared darker than the principal cells.     

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.     

桃小食心虫幼虫的消化道和马氏管的显微结构观察

利用光学显微镜和扫描电子显微镜,在形态学和组织学水平上研究_『桃小食心虫 Carposina sasakii 幼虫消化道和屿氏管的结构.桃小食心虫幼虫消化道由前肠、中肠和后肠组成.前肠细短,肌肉层薄.前肠与中肠交界处有突出的胃盲囊.中肠长且粗大,内有围食膜,肠壁细胞较大,外层为发达的环肌和纵肌.后肠上皮细胞内陷很深.6根念珠状的马氏管位于中、后肠分界处.     

Inhibition of the SAPK/JNK pathway blocks the stimulatory effects of glutamine on fluid secretion by the Malpighian tubules of Rhodnius prolixus

Physiological levels of amino acids such as glutamine, glutamate, aspartate and proline increase the rates of fluid secretion and ion transport by serotonin-stimulated Malpighian tubules (MTs) of Rhodnius prolixus. Here, we examine the proposal that the effects of glutamine are mediated through activation of specific kinases to produce the observed increases in fluid secretion. The glutamine-dependent increase in MT fluid secretion rate was blocked by two chemically unrelated inhibitors of the stress activated protein kinase (SAPK) pathway, SP600125 and dicumarol. Inhibitors of phosphatidyl inositol-3 kinase, p38 mitogen activated protein kinase (MAPK), extracellular-signal regulated kinases and MAPK kinase did not block glutamine's effects on fluid secretion rate when applied at commonly used concentrations. Inhibitors of protein kinase A or C reduced fluid secretion rates of serotonin-stimulated MTs, but did not block the response to glutamine. The glutamine-dependent increase in fluid secretion was also insensitive to cytoskeletal disrupting agents and protein synthesis inhibitors. Results of this study are the first to suggest a role for the SAPK pathway in the control of fluid secretion rates by insect MTs.     

Characterization of transepithelial transport of salicylate by the Malpighian tubules of Drosophila melanogaster and the effects of changes in fluid secretion rate

Abstract.  A radioisotope tracer technique is used to study mechanisms and regulation of transepithelial transport of the plant allelochemical salicylate by the Malpighian tubules of Drosophila melanogaster . Transepithelial transport of salicylate is nearly abolished in Na⁺-free saline, and inhibited by ouabain, low K⁺ or K⁺-free bathing saline. In addition, the carboxylates probenecid, unlabelled salicylate, fluorescein, and p -aminohippuric acid (PAH) significantly inhibit transepithelial transport of salicylate. The sulphonates taurocholate and phenol red also inhibit transepithelial transport of salicylate, whereas amaranth has no effect. Stimulation of fluid secretion by cAMP, cGMP or leucokinin I increases transepithelial transport of salicylate, particularly when the concentration of salicylate in the bathing saline is high. The correlation between the fluid secretion rate and transepithelial transport of salicylate shows that 64% of the changes in salicylate transport can be explained on the basis of changes in fluid secretion rate. The results show that naturally-occurring plant secondary metabolite salicylate is transported into the lumen of the Mapighian tubules of D. melanogaster by a mechanism similar to that previously described for the prototypical organic anions PAH and fluorescein. In addition, the transepithelial transport of salicylate increases in response to increases in fluid secretion rate.