Fluid secretion by in vitro preparations of the Malpighian tubules of the desert locust Schistocerca gregaria

In vitro preparations of locust Malpighian tubules can conveniently be made by a new technique in which the alimentary canal to which the tubules attach is removed from the insect and set up in Ringer's solution under liquid paraffin. Such Malpighian tubules will secrete a fluid iso-osmotic to the bathing fluid at a steady rate of about 1 to 2 nl min^?1 for some hours. The secreted fluid is rich in potassium ions, the lumen is at a potential positive to that of the bathing solution, and the rate of secretion can be controlled by changing the potassium concentration of the bathing fluid. It seems likely, therefore, that an active transport of potassium drives secretion ny locust Malpighian tubules. The secreted fluid contains an elevated concentration of phosphate ions. The Malpighian tubules will secrete at a high rate in a chloride-free phosphate-based solution. The rate of fluid secretion can be increased by treatment with cyclic AMP but 5-hydroxytryptamine has no such effect.     

Anatomy and fine structure of the alimentary canal of the spittlebug Lepyronia coleopterata (L.) (Hemiptera: Cercopoidea)

The alimentary canal of the spittlebug Lepyronia coleopterata (L.) differentiates into esophagus, filter chamber, midgut (conical segment, tubular midgut), and hindgut (ileum, rectum). The filter chamber is composed of the anterior extremity of the midgut, posterior extremity of the midgut, proximal Malpighian tubules, and proximal ileum; it is externally enveloped by a thin cellular sheath and thick muscle layers. The sac-like anterior extremity of the midgut is coiled around by the posterior extremity of the midgut and proximal Malpighian tubules. The tubular midgut is subdivided into an anterior tubular midgut, mid-midgut, posterior tubular midgut, and distal tubular midgut. Four Malpighian tubules run alongside the ileum, and each terminates in a rod closely attached to the rectum. Ultrastructurally, the esophagus is lined with a cuticle and enveloped by circular muscles; its cytoplasm contains virus-like fine granules of high electron-density. The anterior extremity of the midgut consists of two cellular types: (1) thin epithelia with well-developed and regularly arranged microvilli, and (2) large cuboidal cells with short and sparse microvilli. Cells of the posterior extremity of the midgut have regularly arranged microvilli and shallow basal infoldings devoid of mitochondria. Cells of the proximal Malpighian tubule possess concentric granules of different electron-density. The internal proximal ileum lined with a cuticle facing the lumen and contains secretory vesicles in its cytoplasm. Dense and long microvilli at the apical border of the conical segment cells are coated with abundant electron-dense fine granules. Cells of the anterior tubular midgut contain spherical secretory granules, oval secretory vesicles of different size, and autophagic vacuoles. Ferritin-like granules exist in the mid-midgut cells. The posterior tubular midgut consists of two cellular types: 1) cells with shallow and bulb-shaped basal infoldings containing numerous mitochondria, homocentric secretory granules, and fine electron-dense granules, and 2) cells with well-developed basal infoldings and regularly-arranged apical microvilli containing vesicles filled with fine granular materials. Cells of the distal tubular midgut are similar to those of the conical segment, but lack electron-dense fine granules coating the microvilli apex. Filamentous materials coat the microvilli of the conical segment, anterior and posterior extremities of the midgut, which are possibly the perimicrovillar membrane closely related to the nutrient absorption. The lumen of the hindgut is lined with a cuticle, beneath which are cells with poorly-developed infoldings possessing numerous mitochondria. Single-membraned or double-membraned microorganisms exist in the anterior and posterior extremities of the midgut, proximal Malpighian tubule and ileum; these are probably symbiotic.     

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.     

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.     

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.     

The effects of phlorizin,phloretin and ouabain on the reabsorption of glucose by the Malpighian tubules of Locusta migratoria migratorioides

Isolated Malpighian tubules of Locusta reabsorb significant levels of glucose from their lumen back into the bathing fluid (haemolymph). This reabsorption is inhibited by phlorizin, phloretin and ouabain. Both phlorizin and phloretin are found to accumulate in the secreted fluid of the tubules against concentration gradients. Ouabain inhibition is explained in terms of its effect on intracellular Na⁺ concentrations. A hypothetical model of the role that Na⁺ may play in glucose reabsorption is presented as a possible explanation of these observations.     

DNA synthesis during larval development and compensatory growth in the mesonephros of Xenopus laevis.

Autoradiography following tritiated thymidine administration to Xenopus laevis tadpoles of stages 45–48 of larval development has revealed that, as the cells of the mesonephric kidney differentiate during organogenesis, there is a marked decrease in the percentage of cells synthesizing DNA (from 100% at stage 45 to less than 9% at stage 48). In the adult this figure is of the order of 0.1%. This reduced DNA synthetic activity was found to take place in the cells of both the proximal and distal tubules of the nephrons. Special mucous cells which serve as markers of distal tubules were not observed to synthesize DNA after the onset of their differentiation at stage 48 of larval development.Through the partial extirpation of tissue in one kidney of adult Xenopus laevis males, DNA synthesis was reactivated in differentiated cells. The increased DNA synthetic activity following partial unilateral nephrectomy was found to be maximal after 6 days of recovery when 19% of cells synthesize DNA. This increased DNA synthetic activity was found to occur only in the cells of the distal tubules, both mucous and nonmucous cells, while the cells of the proximal tubules did not respond to this reactivation.The apparent inability of proximal tubule cells to synthesize DNA following partial unilateral nephrectomy is discussed.     

The effect of infection with Dirofilaria immitis (dog heartworm) on fluid secretion rates in the Malpighian tubules of the mosquitoes Aedes taeniorhynchus and Anopheles quadrimaculatus

Dirofilaria immitis, the parasitic nematode causing the disease dog heartworm, is transmitted by female mosquitoes. During their development, larval nematodes reside in the cells of the Malpighian tubules of these mosquitoes for approx 13 days. We have examined the effect of the presence of these large intracellular parasites on the main physiological function of the Malpighian tubules, i.e. fluid secretion. Rates of fluid secretion were examined in vitro using both normal and infected tubules of the mosquito species Aedes taeniorhynchus and Anopheles quadrimaculatus. Tubules of A. quadrimaculatus show changes in trasport rate during the reproductive cycle. Those of A. taeniorhynchus do not. Infection with larvae of D. immitis had no effect on the rate of fluid secretion in tubules of A. quadrimaculatus. In A. taeniorhynchus by contrast, the tubules show decline in transport with time following infection. The reduction in transport capacity is proportional to the number of worms infecting the tubule. The present paper and separate ultrastructural studies demonstrate that parallel changes in microvillar ultrastructure and epithelial transport rates occur in response to infection by the parasite. In both species examined, the survival of the mosquitoes and their vector potential are determined by factors other than the transport capacities of the infected Malpighian tubules.     

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.     

Localization and production of immunoreactive endothelin-1 in the trophoblast of human placenta

The elements Na, Cl and K are distributed heterogeneously in the larval cells of the four Malpighian tubules of Drosophila hydei. Elemental gradients are steeper when they are related to the dry weight in comparison to the weight of water. The high contents of Na, Cl and K in the cytoplasm close to the basal plasma membrane differ significantly from the low values found in the neighboring basal labyrinth and in the intermediate cytoplasm. Elemental contents (K, Cl) increase from the intermediate cytoplasm to the apical microvilli and further to the lumen. The K/Na-ratio changes across the cells from 0.9 in the hemolymph to 2 in the basal cytoplasm close to the plasma membrane, to 27 in the microvilli, and 89 in the fluid phase of the lumen. In the lumen, K is accumulated in proteoglycan containing type-II concretions. The water content increases from the distal to the proximal segments of anterior and posterior tubules.     

Stellate cells in the Malpighian tubules of Drosophila hydei and D. melanogaster larvae (Insecta, Diptera)

 The Malpighian tubules of Drosophila hydei and D. melanogaster larvae are composed of two types of cell, principal cells and stellate cells. In the anterior larval Malpighian tubules approximately 26% (D. hydei) and 18% (D. melanogaster), respectively, of all cells are stellate cells. In the larvae of D. melanogaster, the stellate cells are fenestrated and the hemolymph space and tubule lumen are separated only by the basal lamina. Injection of dyes into the hemolymph did not indicate any facilitated transfer of substances through the fenestrated cells. The principal cells of the distal segment are carbonic anhydrase positive indicating transport activity, whereas the stellate cells lack this enzyme. In the stellate cells of the transitional segment, the sodium content is strikingly high in comparison to the neighbouring principal cells and lumen where no sodium was detected. This finding indicates that stellate cells reabsorb sodium as supposed earlier in 1969 by Berridge and Oschman (Tissue Cell 1:247–272). Accepted: 12 February 1999     

Ultrastructure of the malpighian tubules of Schistocerca gregaria

The ultrastructure of the Malpighian tubules of the adult desert locust, Schistocerca gregaria, is described. Male and female adults possess about 233 tubules, which empty proximally into the midgut-ileal region of the alimentary canal by way of 12 ampullae. The tubules vary from 10 mm to 23 mm in length. About one third of them are directed anteriorly, attaching distally at the caeca, while the remainder are directed posteriorly, attaching to other tubules, the rectum or large tracheal trunks adjacent to the hindgut. The Malpighian tubules from all locations examined consist of three ultrastructurally distinct regions: proximal, middle, and distal, referring to their position relative to the midgut. All cell types possess ultrastructural features characteristic of ion transporting tissue, i.e., elaboration of the basal and apical membranes and a close association of these membranes with mitochondria. The distal and proximal segments are short (1.5-1.7 mm) and heavily tracheated, and each is composed of a single, distinct cell type. The middle region is the longest segment of the Malpighian tubule and is composed of two distinct cell types, primary and secondary. Both cell types are binucleate. The more numerous primary cells have large nuclei, contain laminate concretions in membrane-bound vacuoles, and possess large microvilli that contain mitochondria. The secondary cells are smaller and possess smaller nuclei. The microvilli are reduced and lack mitochondria. Secondary cells do not contain laminate concretions. The possible compartmentalization of ion and fluid transport function based on segmentation in the Malpighian tubules is discussed.     

Studies on the morphology and ultrastructure of the Malpighian tubules of Halobiotus crispae Kristensen, 1982 (Eutardigrada)

The excretory and osmoregulatory system of Halobiotus crispae consists of two lateral and one smaller dorsal Malpighian tubules, which empty into the digestive tract in the transition zone of the midgut and rectum. The tubules are identical at the ultrastructural level, and consist of an initial segment with three large cells, a thin transitional distal part lacking a nucleus, and a proximal part with 9–12 nuclei. The initial segment possesses deep basal infoldings and interdigitating, finger-shaped processes of the plasma membrane, large mitochondria and giant nuclei. The distal part is a short section which supports the initial segment. Cellular offshoots from the succeeding proximal part constitute the distal part. The distal and proximal parts contain intercellular canals with concretions of variable size. The exit of the proximal part into the digestive tract is characterized by the presence of microvilli. Correlated with the different stages in the cyclomorphosis of H. crispae , we observed size variation of the Malpighian tubules; thus, pseudosimplex stages have the largest tubules. We present suggestions concerning the physiology of the tubules and compare the Malpighian tubules of Tardigrada with the Malpighian papillae of Protura.     

Segment-specific Ca2+ transport by isolated Malpighian tubules of Drosophila melanogaster: A comparison of larval and adult stages

Haemolymph calcium homeostasis in insects is achieved through the regulation of calcium excretion by Malpighian tubules in two ways: (1) sequestration of calcium within biomineralized granules and (2) secretion of calcium in soluble form within the primary urine. Using the scanning ion-selective electrode technique (SIET), basolateral Ca²⁺ transport was measured at the distal, transitional, main and proximal tubular segments of anterior tubules isolated from both 3rd instar larvae and adults of the fruit fly Drosophila melanogaster. Basolateral Ca²⁺ transport exceeded transepithelial secretion by 800-fold and 11-fold in anterior tubules of larvae and adults, respectively. The magnitude of Ca²⁺ fluxes across the distal tubule of larvae and adults were larger than fluxes across the downstream segments by 10 and 40 times, respectively, indicating a dominant role for the distal segment in whole animal Ca²⁺ regulation. Basolateral Ca²⁺ transport across distal tubules of Drosophila varied throughout the life cycle; Ca²⁺ was released by distal tubules of larvae, taken up by distal tubules of young adults and was released once again by tubules of adults ⩾168 h post-eclosion. In adults and larvae, SIET measurements revealed sites of both Ca²⁺ uptake and Ca²⁺ release across the basolateral surface of the distal segment of the same tubule, indicating that Ca²⁺ transport is bidirectional. Ca²⁺ uptake across the distal segment of tubules of young adults and Ca²⁺ release across the distal segment of tubules of older adults was also suggestive of reversible Ca²⁺ storage. Our results suggest that the distal tubules of D. melanogaster are dynamic calcium stores which allow efficient haemolymph calcium regulation through active Ca²⁺ sequestration during periods of high dietary calcium intake and passive Ca²⁺ release during periods of calcium deficiency.     

The control of Malpighian tubule developmental physiology by 20-hydroxyecdysone and juvenile hormone

The control of developmental changes in Malpighian tubule cell structure and fluid secretion by 20-hydroxyecdysone and juvenile hormone in the skipper butterfly Calpodes ethlius were studied using (1) in vitro tissue culture, (2) in vivo injection and topical application and (3) tubule transplantation experiments. At pupation, 20-hydroxyecdysone initiates cell remodelling and switches off fluid secretion in the Malpighian tubules. Juvenile hormone inhibits these alterations provided that treatment is begun on the first day of the last larval stage. In the pupal stage, 20-hydroxyecdysone triggers the differentiation of adult cell structure which culminates in the renewal of fluid secretion. The results show that 20-hydroxyecdysone and juvenile hormone regulate Malpighian tubule function by altering cell structure and are discussed with respect to the hormonal reprogramming of the Malpighian tubule cells during development.     

The ultrastructure of Malpighian tubules and hindgut of Frankliniella occidentalis (Pergande) (Thysanoptera : Thripidae)

The ultrastructure of the western flower thrips, Frankliniella occidentalis (Pergande) (Order : Thysanoptera), has 4 Malpighian tubules that are free of the intestine as they leave their junction at the pyloric region. The tubules consist of an epithelium with a single type of microvillated cells; proximally, the cells are lined by a thin cuticle. Numerous mitochondria, basal infoldings of the plasma membrane and vesicles with varying densities suggest active transit of fluid in the cell for osmoregulation. Two of the Malpighian tubules are bent posteriorly and closely adhere to the hindgut in the region of the rectal pads where the 2 epithelia are separated only by a basal lamina. The ultrastructure of this region suggests possible fluid reabsorption from the gut lumen.     

Ultrastructure of the larval Malpighian tubules in <Emphasis Type="Italic">Terrobittacus implicatus</Emphasis> (Mecoptera: Bittacidae)

The larvae of Bittacidae, a cosmopolitan family in Mecoptera, have an interesting habit of spraying the body surface with soil through the anus after hatching, and each molts. The fine structure of Malpighian tubules, however, remains largely unknown in the larvae of Bittacidae to date. Here, we studied the ultrastructure of the larval Malpighian tubules in the hangingfly Terrobittacus implicatus (Huang & Hua) using scanning and transmission electron microscopy. The larvae of T. implicatus have six elongate Malpighian tubules at the junction of the midgut and hindgut. The tubule comprises a basal lamina, a single-layered epithelium, and a central lumen. The basal plasma membranes of the epithelial cells are conspicuously infolded and generate a labyrinth. The epithelium consists of two types of cells: large principal cells and scattered stellate cells. Mitochondria and cisterns of rough endoplasmic reticulum are numerous in the principal cells but are sparsely distributed in the stellate cells, indicating that the principal cells are active in transport. On the other hand, spherites are only abundant in the principal cells and are likely associated with the soil-spraying habit of the larvae.     

Ultrastructure of differentiated malpighian tubules from cockroach nymphs during the molting cycle

Differentiated Malpighian tubules of Periplaneta americana nymphs consist of four distinct regions. The distal, middle, and proximal regions are similar to the same regions in adult tubules. However, the transparent portion of the middle region was found to have ultrastructural characteristics different from those of the longer opaque segment of the middle region and the two other tubule regions. This newly distinguished region is called the lower middle region. Transitional zones, areas where cells show characteristics of two adjacent regions, are apparent between the distal and middle regions and between the middle and lower middle regions. The middle region of primary tubules undergoes an increase in autophagic activity and a modification of its basal infoldings and microvilli shortly before each molt. An increase in autophagic activity is also observed in the lower middle region near the time of molting.     

Post-embryonic development of the Malpighian tubules in <Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera) workers: morphology,remodeling, apoptosis,and cell proliferation

The honeybee Apis mellifera has ecological and economic importance; however, it experiences a population decline, perhaps due to exposure to toxic compounds, which are excreted by Malpighian tubules. During metamorphosis of A. mellifera, the Malpighian tubules degenerate and are formed de novo. The objective of this work was to verify the cellular events of the Malpighian tubule renewal in the metamorphosis, which are the gradual steps of cell remodeling, determining different cell types and their roles in the excretory activity in A. mellifera. Immunofluorescence and ultrastructural analyses showed that the cells of the larval Malpighian tubules degenerate by apoptosis and autophagy, and the new Malpighian tubules are formed by cell proliferation. The ultrastructure of the cells in the Malpighian tubules suggest that cellular remodeling only occurs from dark-brown-eyed pupae, indicating the onset of excretion activity in pupal Malpighian tubules. In adult forager workers, two cell types occur in the Malpighian tubules, one with ultrastructural features (abundance of mitochondria, vacuoles, microvilli, and narrow basal labyrinth) for primary urine production and another cell type with dilated basal labyrinth, long microvilli, and absence of spherocrystals, which suggest a role in primary urine re-absorpotion. This study suggests that during the metamorphosis, Malpighian tubules are non-functional until the light-brown-eyed pupae, indicating that A. mellifera may be more vulnerable to toxic compounds at early pupal stages. In addition, cell ultrastructure suggests that the Malpighian tubules may be functional from dark-brown-eyed pupae and acquire greater complexity in the forager worker bee.