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.     

The role of adenosine 3':5'-cyclic monophosphate in relation to the diuretic hormone of Rhodnius prolixus.

The relationship between diuretic hormone (DH) and adenosine 3′:5′-cyclic monophosphate (cyclic AMP) in Rhodnius Malpighian tubules has been investigated. Direct measurement of cyclic AMP levels during stimulation of the tubules by DH supports the view that cyclic AMP is a ‘second messenger’ in this system.Also, the activity of endogenous cyclic AMP phosphodiesterase and its inhibition by theophylline has been investigated briefly. Certain other 3′:5′-cyclic nucleotides have been examined for diuretic activity on Rhodnius Malpighian tubules.     

Number of Malpighian Tubules in Larvae and Adults of Stingless Bees (Hymenoptera: Apidae) from Amazonia

The number of Malpighian tubules in larvae and adults of bees is variable. Larvae of Apis mellifera L. have four Malpighian tubules, while adults have 100 tubules. In stingless bees, this number varies from four to eight. The objectives of this study were to provide characteristics of the Malpighian tubules as well as to quantify their number in larvae and adults of six species of Meliponinae, Melipona seminigra merrillae Cockerell, Melipona compressipes manaosensis Schwarz, Melipona rufiventris Lepeletier, Scaptotrigona Moure, Frieseomelitta Ihering, and Trigona williana Friese. Malpighian tubules were dissected from larvae and adults, measured, quantified, and maintained in microtubes with Dietrich??s solution. The numbers of Malpighian tubules were constant only for larvae of M. rufiventris (four and eight) and Scaptotrigona sp. (four). The most frequent number of tubules in the Melipona group was seven and eight in larvae, and 70 and 90 in adults. In the Trigona group were four and 20 to 40, for larvae and adults, respectively. The results showed differences in the number of Malpighian tubules among the species analyzed and also between the larvae and adults of the same species. Despite the variation observed, species of the group Melipona always have a larger number and longer Malpighian tubules in both larvae and adults as compared to the Trigona group, which may indicate an evolutionary trend of differentiation between these groups.     

Gregarina hylobii and Ophryocystis hylobii n. sp. (ophryocystidae,neogregarinida) parasitizing Hylobius abietis (Curculionidae,coleoptera)

Two sporozoan parasites, Gregarina hylobii and Ophryocystis hylobii n. sp., parasitizing the adults of natural populations of Hylobius abietis are described. The gregarine G. hylobii, a parasite of the gut of H. abietis, has been reexamined and its complete life cycle, including the solitary gamonts, gametocysts, and sporoducts is described. The life cycle of the neogregarine O. hylobii infecting the Malpighian tubules of host animals examined at light and electron microscope levels is discussed. Some data on the prevalence of infections and host-parasite relationships are also discussed.     

More on the life cycle of Malamoeba scolyti (Amoebidae: Sarcomastigophora) parasitizing: the bark beetle Dryocoetes autographus (Scolytidae,Coleoptera)

Some new developmental features previously unreported in, the life- cycle of an amoeba Malamoeba scolyti, which cause cysts in Malpighian tubules of the bark beetle Dryocoetes autographus, are described by means of light, phase-contrast, Nomarski interference, and electron microscopy. Two developmental phases are presumed to take place in the life cycle of M. scolyti: a phase with large multinucleate amoebae (trophozoites) developing in the gut and caecum of the host, and a phase (parasitic phase) causing cysts in the Malpighian tubules. Some data on the host-parasite relationship and rate of infection are also discussed.     

Structural and ultrastructural alterations of Malpighian tubules of Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) larvae infected with different Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) recombinant viruses

Malpighian tubules constitute the main excretion organ of insects. Infection by egt⁻ recombinant AcMNPV baculovirus in lepidopteran larvae promotes early degeneration of these structures, which has been correlated with earlier death of the host. However, no trace of viral infection has been detected in that tissue. We constructed two AgMNPV recombinants with the egfp gene under control of the hsp70 promoter, one being egt⁻, and used another two recombinants (one egt⁻) containing the lacZ gene. Morphological alterations in the tubules were analyzed by light and electron microscopies. Bioassays were conducted to compare the pathogenicity of recombinants. Results showed progressive presence of marker proteins and tissue degeneration without signals of infection in the tissue. Morphological and bioassay results showed increased pathogenicity for lacZ-containing recombinants compared to the egfp ones; as for egt⁻ viruses, we noted higher intensity and earlier onset of alterations. The absence of infection led us to believe that Malpighian tubules degeneration is provoked initially by the death of tracheal cells attached to the tubules and later, by the death of Malpighian tubule cells themselves. Tubule cell death might be due to oncosis and apoptosis, which may be activated by depletion of energy reserves and by accumulation of marker proteins, respectively. Absence of the egt gene may be leading to a higher energetic expense due to molting, thus aggravating tubule cell death, resulting in faster death of host.     

Histopathology of secondary infections of Malpighamoeba locustae (Protozoa,Amoebidae) in the desert locust, Schistocerca gregaria (Orthoptera,Acrididae)

Schistocerca gregaria, the desert locust, harbors the protozoan parasite Malpighamoeba locustae. Twelve to 16 days after infection, trophozoites begin to multiply rapidly inside the Malpighian tubules of the gut. Swelling and rupture of the Malpighian tubules leads to the release of large numbers of cysts and trophozoites into the hemocoel. The classic insect defense response results in these cysts and trophozoites becoming encapsulated by the hemocytes of the host. Hemocytes of the phagocytic type become attached to, and lodged between, a variety of tissues and organs of the locust and black hemocytic capsules are produced. The extent to which the different tissues are involved is graded. Some tissues are completely blackened and encapsulated by masses of hemocytes but others are so lightly affected that the small specks of blackened pigment they lay down are discernible only on close examination. The trophozoites themselves do not divide outside the Malpighian tubules. The graded response in the host tissues is related to (1) the presence of sheets and lobes of fat body and (2) the presence of phagocytic hemocytes.     

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.     

Partial characterization of N-acetyltransferase activity from cerebral ganglia and malpighian tubules of Periplaneta americana

Octopamine-N-acetyltransferase (NAT) activity from Malpighian tubules and cerebral ganglia of Periplaneta americana was studied using high performance liquid chromatography with coulometric electrochemical detection. The enzyme from these tissues is highly soluble and temperature-resistant with maximal activity demonstrated at 50°C. The pH profiles of enzyme activity from Malpighian tubules and cerebral ganglia differ markedly although, in both tissues, strong activity is evident under alkaline conditions. Kinetic analyses indicate that the enzyme present in Malpighian tubules has a K_m for octopamine of 0.40 mM with a maximum velocity of 37.9 nmol N-acetyl-p-octopamine (N-acOA) produced/mg protein/min; the equivalent values for acetyl CoA were determined as 0.32 mM and 36.9 nmol N-acOA/mg protein/min. NAT from cerebral ganglia showed K_ms for octopamine and acetyl CoA of 0.33 and 0.19 mM, respectively, and the maximum velocity with octopamine was 6.8 nmol N-acOA/mg protein/min. Analysis of NAT activity from a variety of cockroach tissues indicates that the enzyme is widely distributed, with the gut and associated Malpighian tubules, conglobate gland and salivary glands showing the greatest activity.     

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.     

Relationship of Na+-K+-activated ATPase to fluid production by Malpighian tubules of Locusta migratoria.

The presence of a Na⁺K⁺-activated, Mg²⁺-dependent ATPase (E.C. 3.6.1.3) has been demonstrated in microsomal preparations from the Malpighian tubules of Locusta. The effects of sodium and potassium ions, and different concentrations of ouabain, have been studied in relation to the activity of this enzyme and the ability of in vitro Malpighian tubule preparations to secrete fluid. From these studies it seems highly likely that a Na⁺K⁺ activated ATPase ‘pump’ is involved in fluid transport across the walls of the tubules.     

Scalariform junctions in the malpighian tubules of the insects Rhodnius prolixus (Hemiptera: Reduviidae) and Aedes taeniorhynchus (Diptera: Culicidae)

The ultrastructure of scalariform junctions in the Malpighian tubules of the hemipteran Rhodnius prolixus and the dipteran Aedes taeniorhynchus is described. Both autocellular and intercellular scalariform junctions are illustrated. This is the first report of scalariform junctions in the Malpighian tubules of a dipteran. When combined with previous observations by other authors, the presence of scalariform junctions has now been reported in the Malpighian tubules of insects from five orders, including ametabolous, hemimetabolous, and holometabolous forms. The cell types in which scalariform junctions were found in R. prolixus and A. taeniorhynchus differ in the direction of ion and fluid transport. The cells share the capacity to transport KCl. These same cells also possess morphological features promoting close associations of mitochondria and plasma membranes in the apical region of the cell. The possible role of scalariform junctions is discussed in light of these observations.     

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.     

Morphology of the Phytophagous bug Platyscytus decempunctatus (Carvalho) (Heteroptera: Miridae)

Platyscytus decempunctatus Carvalho is a small mirid living throughout its life cicle plant Solanum cernuum Vell. (Solanaceae). The internal morphology of P. decempunctatus was studied and found that the female has a telotrophic ovary with three ovarioles in the right ovary and five in the left one. In the male reproductive trait there is a well developed tubular accessory gland. The phytophagous feeding habitat of P. decempunctatus was confirmed by the observations of chlorophyll pigments in the gut of all dissected specimens. The tubular salivary glands and the Malpighian tubules were also discussed.     

Expression patterns of genes encoding proteins with peritrophin A domains and protein localization in Mamestra configurata

Genes encoding three proteins (McPPAD1-3) with peritrophin A chitin-binding domains (PADs) were identified from a Mamestra configurata larval midgut cDNA library. In addition to midgut, McPPAD1-3 and a previously identified gene encoding the peritrophin, McPM1, were expressed in foregut, hindgut, Malpighian tubules, tracheae, fat body and cuticle; however, the corresponding McPPAD proteins exhibited different localization patterns. McPPAD1 was restricted to the digestive tract and Malpighian tubules, McPPAD2 to Malpighian tubules, and McPPAD3 to the foregut, midgut, hindgut, tracheae and cuticle. Protein fold recognition analysis using tachycitin as a guide structure modelled the McPPAD1 PADs, but not McPPAD2 or McPPAD3 PADs. The McPPAD1 PADs were predicted to contain three anti-parallel β-sheets and a hevein-like fold that form a chitin-binding pocket containing two hydrophobic R-groups in a sandwich-like orientation.     

Two New Species of Nosema (Microsporida: Nosematidae) from the Mexican Bean Beetle Epilachna varivestis (Coleoptera: Coccinellidae)1,2

Two new species of the genus Nosema (Microsporida: Nosematidae) are described from the Mexican bean beetle, Epilachna varivestis (Coleoptera: Coccinellidae) and their life cycle stages studied by light and electron microscopy. Both species are monomorphic and disporous: they develop in direct contact with the cytoplasm of host cells and the nuclei of all stages are diplokaryotic. The more virulent species produces systemic infections most extensively in the adipose tissue, muscles, and Malpighian tubules of larvae and also invades the reproductive tissues of adult beetles. During merogonic development, it forms chains of diplokaryotic meronts. The fine structure of the sporoblast nuclei shows clumped material in the pole of each nucleus opposite their common plane of apposition. Spores are straight to slightly curved and ovocylindrical in shape and they measure 5.3 ± 0.13 × 2.1 ± 0.03 μm. The less virulent species also invades most host tissues but does not develop in the midgut epithelium; the Malpighian tubules are the principal site of its development and it also invades the ovaries and testes of adult beetles. Merogony occurs exclusively as the result of binary fission of diplokaryotic meronts. The plasmalemma of the meronts is covered with a thin deposit of exospore material upon which are located closely packed tubules that encircle the body transversely. A thickened deposit of exospore material on the surface of the diplokaryotic sporonts later obscures these tubules. Other tubules occur free in the host cell cytoplasm or attached to the plasmalemma of meronts and sporonts. Secretory granules also occur free or in chains in the host cytoplasm and are probably produced from the surface of the sporoblasts. Sporoblasts also contain an unusual cup-shaped organelle associated with a dense body, which is apparently involved in the formation of the polar tube and its associated organelles in the anterior part of the spore. Spores are ellipsoidal to slightly pyriform and measure 4.7 ± 0.06 × 2.6 ± 0.03 μm.     

Cloning and functional characterization of inward-rectifying potassium (Kir) channels from Malpighian tubules of the mosquito Aedes aegypti

Inward-rectifying K⁺ (Kir) channels play critical physiological roles in a variety of vertebrate cells/tissues, including the regulation of membrane potential in nerve and muscle, and the transepithelial transport of ions in osmoregulatory epithelia, such as kidneys and gills. It remains to be determined whether Kir channels play similar physiological roles in insects. In the present study, we sought to 1) clone the cDNAs of Kir channel subunits expressed in the renal (Malpighian) tubules of the mosquito Aedes aegypti, and 2) characterize the electrophysiological properties of the cloned Kir subunits when expressed heterologously in oocytes of Xenopus laevis. Here, we reveal that three Kir subunits are expressed abundantly in Aedes Malpighian tubules (AeKir1, AeKir2B, and AeKir3); each of their full-length cDNAs was cloned. Heterologous expression of the AeKir1 or the AeKir2B subunits in Xenopus oocytes elicits inward-rectifying K⁺ currents that are blocked by barium. Relative to the AeKir2B-expressing oocytes, the AeKir1-expressing oocytes 1) produce larger macroscopic currents, and 2) exhibit a modulation of their conductive properties by extracellular Na⁺. Attempts to functionally characterize the AeKir3 subunit in Xenopus oocytes were unsuccessful. Lastly, we show that in isolated Aedes Malpighian tubules, the cation permeability sequence of the basolateral membrane of principal cells (Tl⁺ > K⁺ > Rb⁺ > NH₄⁺) is consistent with the presence of functional Kir channels. We conclude that in Aedes Malpighian tubules, Kir channels contribute to the majority of the barium-sensitive transepithelial transport of K⁺.     

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

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

Cryptonephric malpighian tubule system in a dipteran larva,the New Zealand glow-worm, Arachnocampa luminosa (Diptera: Mycetophilidae): A structural study

The Malpighian tubules of the glow-worm are divided into four morphologically distinct regions, each composed of a different cell type. Part 3 of the Malpighian tubules of A. luminosa is intimately bound to the rectum by a layer of fat body. This association of the tubules with the hindgut is referred to as a cryptonephric system. This type of arrangement has been described in some Coleoptera and the larvae of most Lepidoptera but has never before been reported in the Diptera. In the glow-worm the cryptonephric tubules themselves are small, and adjacent to the fat body the epithelial cells are modified to form very thin windows or ‘leptophragmata’ (Lison, 1937). The main epithelial cells exhibit features characteristic of highly active, secretory Malpighian tubule cells. The high density of mitochondria and their association with all the microvilli is indicative of a highly active secretory cell. The high concentration of glycogen in these cells and their intimate association with the hindgut suggest that they may, in addition, have a nutrient absorptive function. The role of the cryptonephric rectal complex in the glow-worm is discussed in the light of present knowledge gained from previous studies of coleopteran and larval lepidopteran cryptonephric systems. On structural grounds a model is proposed for the regulation of the ionic environment of the rectum, and the uptake and metabolism of organic material from the rectal lumen by this cryptonephric complex.