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.     

Cell proliferation and rearrangement in the development of the Malpighian tubules of the hemipteran, Rhodnius prolixus

The pattern of cell activities resulting in the generation of a simple tubular epithelium, the Malpighian tubules of Rhodnius prolixus, is examined during embryogenesis. The anlage of the tubules is shown to be of ectodermal origin. An evolving pattern of cycling cells in the primordia is revealed using the immunocytochemical localization of a substituted nucleotide, BUdR. A cell unique to each tubule is shown not to enter the cell cycle but to be required for the proliferation of the remaining cells in the tubules. Furthermore, the activity of this cell imposes on each tubule a clear proximo-distal axis.     

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.     

Ultrastructure of osmoregulatory organs in larvae of the brackish-water mosquito,Culiseta inornata (Williston)

The ultrastructure of the Malpighian tubules, ileum, rectum, anal canal, and anal papillae of larvae of the mosquito Culiseta inornata was examined. The Malpighian tubules, rectum, and anal papillae have many of the ultrastructural features characteristic of ion transport tissues, i.e., elaboration of the basal and apical membranes and a close association of these membranes with mitochondria. The Malpighian tubules possess two cell types, primary and stellate. The larval rectum of C. inornata is composed of a single segment containing a homogenous population of cells. In this respect, the larval rectum of C. inornata is distinct from that of saline-water species of Aedes. The cells in the larval rectum of C. inornata, however, closely resemble those of one cell type, the anterior rectal cells, of the saline-water mosquito Aedes campestris with regard to cell and nuclear size, the percentage of the cell occupied by apical folds, and mitochondrial density and distribution. No similarities can be found between the rectum of C. inornata and the posterior segment of the saline-water Aedes, which functions as a salt gland. On this basis, we have postulated that the rectum of C. inornata does not function as a site of hyperosmotic fluid secretion. The ultrastructure of the anal papillae of C. inornata is consistent with a role in ion transport. The significance of these findings to comparative aspects of osmoregulatory strategies in mosquito larvae is discussed.     

The ultrastructure of the larval malpighian tubules of a saline-water mosquito

The larval Malpighian tubules of the saline-water mosquito Aedes taeniorhynchus were examined using light and electron microscopy. The tubules contain two cell types: primary cells and stellate cells. Primary cells are characterized by their size (70 μm × 70 μm × 10 μm) and an abundance of intracellular membranebound crystals. Two types of microvilli are found on the luminal surface of the primary cells: (1) small microvilli containing core microfilaments and extensions of endoplasmic reticulum, and (2) larger microvilli (≈3 μm in length) which in addition to the above components contain a mitochondrion along their entire length. Both microvillar types have abundant knobs lining the cytoplasmic surface of the microvillar membrane. These knobs, which are often found in insect ion transporting tissues, have been termed ‘portasomes’ by Harvey (1980). The possible role of these structures in ion transport and mitochondrial positioning is discussed. The stellate cells are much smaller than the primary cells, and lack intracellular crystals. Their microvilli are smaller as well (≈0.6 μm in length) and contain no endoplasmic reticulum. mitochondria or knobs. The cells types found in the saline-water mosquito larva, Aedes taeniorhynchus, are identical to those found in Aedes aegypti, indicating that the unique capacity of saline-water mosquito larvae to transport Mg²⁺ and SO₄|post|staggered|2− is not associated with the presence of an additional cell type.     

The ultrastructure of the anal vesicle of the parasitoid,Microplitis croceipes (Hymenoptera:Braconidae)

The fine structural characteristics of epithelial cells of the anal vesicle in the hymenopteran parasitoid, Microplitis croceipes (Cresson), are similar to those of transport cells. Apical and basal infoldings, an abundance of mitochondria, ribosomes, rough endoplasmic reticulum, Golgi complexes and pinocytotic vesicles all indicate a transport function for these epithelial cells. The medial portions of both Malpighian tubules located within the anal vesicle also were examined and on the basis of morphology appear to be active. These observations support earlier physiological data which indicate that the anal vesicle functions in absorption of nutrients and excretion.     

DNA replication in binucleate cells of the Malpighian tubules of Hemipteran insects

The cells of Malpighian tubules of the Hemipteran blood-sucking insect, Rhodnius prolixus, are binucleate. The cells grow without division and, at each larval moult, the DNA content of the nuclei doubles. At the final moult to the adult, however, the DNA content does not change, even though the tubules grow considerably thereafter. In contrast, the DNA content of the tubule nuclei of two other Heteropteran Hemipteran insects, Dysdercus and Oncopeltus, doubles at every moult, including the final one to the adult. If extra larval moults are induced in Rhodnius, by treatment with juvenile hormone, DNA doubling is induced at each such supernumerary larval moult. Shortly after the DNA content increases in Rhodnius tubules, the chromosomes can be seen in a condensed state; presumably, therefore, DNA replication is achieved by endomitosis. Both before this DNA doubling, and within a day after, multiple nucleoli are prominent and appear actively engaged in producing ribosomal precursors. In fed fourth stage Rhodnius, the DNA content of the tubule cell nuclei increases 5–6 days after the blood meal. Neither the rate of fluid secretion that can be induced by stimulation nor the rate of transport of p-aminohippuric acid show any change at the time of DNA replication nor in the remaining days before ecdysis to the fifth stage. Malpighian tubule cell growth without division is thus well adapted to providing, without interruption, for the excretory needs of the growing insect.     

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.     

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     

The distribution of cell types in the malpighian tubules of Aedes taeniorhynchus (Wiedemann) (Diptera : Culicidae)

We have developed a histological technique that allows all of the cells in the malpighian tubules of Aedes taeniorhynchus (Diptera : Culicidae) and a few other mosquitoes to be counted and identified to cell type. We used this technique to examine the number and distribution of primary and stellate cells. Tubules, taken from the 4th instar larvae, pupae, adult males, and adult females, were examined. The mosquitoes were reared as larvae in either 10% or 100% seawater. In all cases examined, the total number of cells composing a tubule was somewhat variable, but no correlation between cell number and developmental stage, environmental salinity, or adult sex was observed. Under all conditions examined, stellate cells compose a fairly constant 16–18% of the total cell population of the tubule. Stellate cells were absent from the tubules in a region near the point of attachment to the midgut. Similar distribution ratios of primary and stellate cells were observed in the tubules of Aedes aegypti, Culex tansalis, and Culiseta inornata. The significance of these findings to a previously proposed tole for stèllate cells in ion transport is discussed.     

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.     

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.     

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     

Fine structure of the rectum in cockroaches (Dictyoptera): General organization and intercellular junctions

The organization of the rectal pads is described in cockroaches belonging to the Groups Blattoidea (Periplaneta americana, Blatta orientalis) and Blaberoidea (Supella supellectilium, Blaberus craniifer). In the Blattoidea, each pad is composed of two layers (principal and basal cells) and is surrounded by very narrow junctional cells supporting the sclerotized cuticle of the pad frame; basally, the junctional cells abut on to the basal cells. In the Blaberoidea, the basal cell layer is discontinuous, the basal cells being interspersed between extensions of the junctional cells beneath the pad. The ultrastructural features of each cell type is described, with special reference to the intercellular junctions, which exhibit unusual complexity. Four types of junction are recognized: desmosomes (belt and spot desmosomes), gap junctions, septate junctions and scalariform (ladder-like) junctions. The last are usually closely associated with mitochondria, forming mitochondrial-scalariform junction complexes (MS). The distribution of these junctions is examined in relation to the partitioning of extracellular spaces, and to the problem of fluid transport.     

Cell junctions and other membrane specializations in the ocellus of the wasp,Paravespula Germanica L. (Hymenoptera : Vespidae)

Five types of cell contacts and other membrane specializations were found in the ocellus of the adult wasp, Paravespula germanica L. (Hymenoptera : Vespidae), based on freeze-fracture replicas and thin sections.Septate junctions alongside small gap junctions are present between iris cells and between corneagenous cells. Gap junctions are sometimes observed between glial cell processes. Photoreceptor cells and glial cells are frequently connected by scalariform junctions. Tight junction-like structures are found on receptor-cell membranes near rhabdomeric microvilli. Desmosomes are widespread in the ocellus, connecting iris cells, corneagenous cells, receptor cells, and glial cell processes. Desmosomes are found next to septate junctions.Glial membranes connected to receptor cells have a non-junctional type of membrane specializations, consisting of intramembraneous particles arranged in a rhombic pattern. Interestingly, both particle arrays and scalariform junctions are often adjacent to each other. Furthermore, a conspicuous modification of the cell surface in freeze cleaved cells is seen between adjacent glial cells intermediating two receptor cells.     

Intracellular melanization of the larvae of Dirofilaria immitis in the malpighian tubules of the mosquito, Aedes sollicitans

Frequent melanization of larvae of the nematode Dirofilaria immitis parasitizing the Malpighian tubules of the mosquito, Aedes sollicitans, has been observed. Melanized and nonmelanized larvae in the Malpighian tubules were examined using light and electron microscopy. The results indicate that the pattern of melanin deposition and the ultrastructural characteristics of the pigment around the worms are identical to that observed on nematodes which have undergone humoral melanization in other dipteran insects. In the Malpighian tubules, no contact between the intracellular melanized nematodes and the hemolymph or hemocytes was observed. The results suggest that the Malpighian tubules of this species of mosquito are capable of inducing a melanotic response to invading nematode parasites. It is proposed that this is an example of “humoral” melanization at an intracellular site.     

Removal of insect basal laminae using elastase

We have used the enzyme elastase to remove the basal lamina of epithelia from two insects: the upper Malpighian tubules of Rhodnius prolixus and imaginai discs of Drosophila melanogaster. Removal of the basal lamina was confirmed using scanning and transmission electron microscopy. Use of the technique on the Malphighian tubules of Rhodnius reveals for the first time the three-dimensional organization of the circumferential folds of the basal plasma membrane. Elastase is much more effective in removing the basal lamina than are the enzymes hyaluronidase, collagenase, and chymotrypsin, either alone or in combination. Following elastase treatment, cells of the Malpighian tubules dissociate with only mild mechanical agitation into single, viable cells. Treatment with elastase removes the basal laminae of imaginai discs of Drosophila and accelerates evagination as has been previously described for trypsin. To obtain single cell preparations from elastase-treated imaginai discs, mechanical stirring in Ringer low in Ca²⁺ was required. In addition to its usefulness in cell isolation, elastase treatment allows examination of the effect of removal of basal laminae on the physiology and development of insect epithelia.     

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.     

Genetic Control of Development of Malpighian Tubules in Drosophila melanogaster

Malpighian tubules of insects are a functional analog of mammalian kidneys and serve as a classical model for studying the structure and functions of transport epithelium. The review contains the data on structural organization, functioning, and formation of the Malpighian tubules during embryogenesis in Drosophila melanogaster. Various systems of genes are described that control the program of development of the renal (Malpighian) tubules in D. melanogaster. A special attention is paid to the ways of signal transduction and factors involved in cell differentiation, proliferation, and morphological transformation during development of the Malpighian tubules. Evolutionarily conservative genetic systems are considered that are involved in the control of development of both the renal epithelium ofDrosophila and mammalian kidneys. A relationship was noted between the disturbed balance of genetic material and congenital defects of the human excretory system.     

Genetic and functional analysis of tryptophan transport in Malpighian tubules of Drosophila

Dissected Malpighian tubules from wild type and the eye color mutant white of Drosophila were compared with respect to their abilities to transport tryptophan and kynurenine into tubule cells. It was determined that mutation at white greatly impairs the ability of Malpighian tubule cells to take up tryptophan. Functional studies on the extracellular spaces and ultrastructural observations indicated no differences in these respects between wild type and white tubules. It is consistent with several observations that much of the tryptophan associated with white exists in the intercellular spaces. Furthermore, the uptake of tryptophan by the w ⁺ system of wild type tubules is inhibited by the analogue 5-methyl-tryptophan. However, the incorporation of radioactive tryptophan into protein in tubule cells from wild type and white occurs at the same rates and is not affected by 5-methyl-tryptophan. Therefore, it is apparent that Malpighian tubules have a transport system that enables entry of tryptophan into a cellular pool and that this cellular pool is initially independent of the tryptophan pool used for protein synthesis. The mutant white lacks this transport system. From these studies and others it appears that compartmentalization of cellular pools may be brought about via the utilization of specific membrane transport systems.