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.     

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.     

Transport defects as the physiological basis for eye color mutants of Drosophila melanogaster

Kynurenine-H ³ transport and conversion to 3-hydroxykynurenine were studied in organ culture using the Malpighian tubules and developing eyes from wild type and the eye color mutants w, st, 1td, ca, and cn of Drosophila melanogaster. Malpighian tubules from wild type have the ability to concentrate kynurenine and convert it to 3-hydroxykynurenine. The tubules from w, st, 1td, and ca are deficient in the ability to transport kynurenine, as are the eyes of the mutants w, st, and 1td. This defect in kynurenine transport provides a physiological explanation for the phenotypic properties of the mutants. The relationship of these measurements to previous observations on these eye color mutants is discussed and the transport defect hypothesis is consistently supported. We have concluded that several of the eye color mutants in Drosophila are transport mutants.     

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.     

Molecular characteristic of stable and unstable <Emphasis Type="Italic">white</Emphasis> gene alleles in highly mutable lines from natural <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> populations

Mutations in the white locus emerged in highly mutable isofemale Drosophila melanogaster lines from the populations of Novosibirsk 2013 (NS3 line), Nalchik 2014 (N119 line), and Sakhalin Island 2014 (S46 line). A single white-eyed male found in the NS3 line was sterile. Phenotypically mutant derivatives (white gene alleles) differing in eye color (pure white, different shades of yellow (honey), orange (apricot), cherry, and red (wild type)) emerged during the N119 and S46 line breeding in the laboratory. Molecular genetic study of the structure of wild type white locus in initial lines and white-mutant derivatives de novo emerging from them, as well as other white lines from the fund of the Laboratory of Population Genetics of the Institute of Cytology and Genetics (Siberian Branch, Russian Academy of Sciences), was conducted. The pairs of primers flanking different white gene regions were selected. Six such pairs overlapped the coding part of the gene. Molecular genetic analysis demonstrated that most DNA defects were limited to the region which includes the first exon (34 lines). Among them, four mutant events were accompanied by an insertion of DNA fragments of approximately 800 bp; one mutation event was accompanied by a deletion of approximately 200 bp; in 29 cases, no PCR product was obtained (this can indicate that as a minimum one of the primer binding sites is damaged). The inserted DNA fragments have no homology with known D. melanogaster sequences presented in the NCBI database. The complete white gene deletion with the manifestation of mutant “white eyes” phenotype was registered in four cases (and only in the N119 line derivatives). Normal PCR product was obtained in 22 cases for all six DNA fragments. Among them, there are both alleles phenotypically mutant by the eye color (white, cherry, or orange) and revertants to the wild type (red). The abundance of defects in the beginning of the gene can indicate a multiplicity of mobile genetic element insertion sites in this part of the white gene in D. melanogaster.     

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.     

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.     

The corticotropin-releasing factor-like diuretic hormone 44 (DH44) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster

Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in insects, and are under neuroendocrine control by multiple neuropeptides secreted by identified neurons. Here, we demonstrate roles for CRF-like diuretic hormone 44 (DH₄₄) and Drosophila melanogaster kinin (Drome-kinin, DK) in desiccation and starvation tolerance.Gene expression and labelled DH₄₄ ligand binding data, as well as highly selective knockdowns and/or neuronal ablations of DH₄₄ in neurons of the pars intercerebralis and DH₄₄ receptor (DH₄₄-R2) in Malpighian tubule principal cells, indicate that suppression of DH₄₄ signalling improves desiccation tolerance of the intact fly.Drome-kinin receptor, encoded by the leucokinin receptor gene, LKR, is expressed in DH₄₄ neurons as well as in stellate cells of the Malpighian tubules. LKR knockdown in DH₄₄-expressing neurons reduces Malpighian tubule-specific LKR, suggesting interactions between DH₄₄ and LK signalling pathways.Finally, although a role for DK in desiccation tolerance was not defined, we demonstrate a novel role for Malpighian tubule cell-specific LKR in starvation tolerance. Starvation increases gene expression of epithelial LKR. Also, Malpighian tubule stellate cell-specific knockdown of LKR significantly reduced starvation tolerance, demonstrating a role for neuropeptide signalling during starvation stress.     

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.     

Free tryptophan pool and tryptophan biosynthetic enzymes in Saccharomyces cerevisiae

The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase) have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.The tryptophan pool of wild type cells growing in minimal medium is 0.07 mole per g dry weight. Addition of anthranilate, indole or tryptophan to the medium produces a fifteen- to forty-fold increase in tryptophan pool, but causes no repression of the biosynthetic enzymes. Inclusion of 5-methyltryptophan in the growth medium causes a reduction in growth rate and a derepression of the biosynthetic enzymes, and this is shown here not to be correlated with a decrease in the free tryptophan pool.Mutants with an altered anthranilate synthase showing decreased sensitivity to inhibition by l-tryptophan or by the analogue dl-5-methyltryptophan have a tryptophan pool far higher than the wild type strain, but no repression of indoleglycerolphosphate synthase was observed. Mutants with an anthranilate synthase more sensitive to tryptophan inhibition show a slightly reduced tryptophan pool, but no derepression of indoleglycerolphosphate synthase was found.A mutant with constitutively derepressed levels of the biosynthetic enzymes shows a considerably increased tryptophan pool. Addition of 5-methyltryptophan to the growth medium of non-derepressible mutants causes a decrease in growth rate accompanied by a decrease in the tryptophan pool.Abbreviations CDRP 1-(o-carboxyphenylamino)-1-deoxyribulosephosphate - paba paraaminobenzoic acid - PRA N-(5-phosphoribosyl)-anthranilate - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for corresponding tryptophan biosynthetic enzymes     

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.     

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.     

Interactions between epithelial nitric oxide signaling and phosphodiesterase activity in Drosophila

Signaling by nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cGMP) modulates fluid transport in Drosophila melanogaster. Expression of an inducible transgene encoding Drosophila NO synthase (dNOS) increases both NOS activity in Malpighian (renal) tubules and DNOS protein in both type I (principal) and type II (stellate) cells. However, cGMP content is increased only in principal cells. DNOS overexpression results in elevated basal rates of fluid transport in the presence of the phosphodiesterase (PDE) inhibitor, Zaprinast. Direct assay of tubule cGMP-hydrolyzing phosphodiesterase (cG-PDE) activity in wild-type and dNOS transgenic lines shows that cG-PDE activity is Zaprinast sensitive and is elevated upon dNOS induction. Zaprinast treatment increases cGMP content in tubules, particularly at the apical regions of principal cells, suggesting localization of Zaprinast-sensitive cG-PDE to these areas. Potential cross talk between activated NO/cGMP and calcium signaling was assessed in vivo with a targeted aequorin transgene. Activated DNOS signaling alone does not modify either neuropeptide (CAP_2b)- or cGMP-induced increases in cytosolic calcium levels. However, in the presence of Zaprinast, both CAP_2b-and cGMP-stimulated calcium levels are potentiated upon DNOS overexpression. Use of the calcium channel blocker, verapamil, abolishes the Zaprinast-induced transport phenotype in dNOS-overexpressing tubules. Molecular genetic intervention in the NO/cGMP signaling pathway has uncovered a pivotal role for cell-specific cG-PDE in regulating the poise of the fluid transporting Malpighian tubule via direct effects on intracellular cGMP concentration and localization and via interactions with calcium signaling mechanisms. Malpighian tubule; cGMP; calcium; aequorin; CNG channel     

Ultrastructural changes in the gut and the Malpighian tubules of Epilachna varivestis after application of the new antibiotic Nikkomycin involve an osmoregulatory defect

The effect of the antibiotic Nikkomycin was investigated on the Malpighian tubules and the gut of fourth-instar larvae of the Mexican bean beetle, Epilachna varivestis. Within the Malpighian tubules, three different stages in cell alterations can be recognized. A stage of increased activity (Stage A), and two stages of dedifferentiation (Stages B and C) which are distinguishible by characteristic mitochondrial morphology. In Stage C individuals, when Malpighian tubule function stops entirely, alterations in the midgut take place, that are signs of increased activity. Measurements of hemolymph osmotic pressure showed that there is a considerable increase to a higher level which is maintained. Compared with the ultrastructural data, the regulation of osmotic pressure on a higher level may, in part, be the result of compensation for the failure of Malpighian tubule function by the midgut.     

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.