Developmental changes in Malpighian tubule fluid transport

Developmental changes in Malpighian tubule fluid transport were studied using in vitro and in situ preparations from timed larval, pupal and adult skipper butterflies (Calpodes ethlius). The ability to transport fluid and the rate of fluid secretion depend on the developmental stage and the physiological state. Larval tubules are permanently switched on, do not require a diuretic hormone and transport fluid at an increasing rate as the larvae feed and grow. Fluid transport continues at larval-larval moults but is switched off 24 hr before pupal ecdysis. No secretory activity occurs during the first half of the pupal stage when the tubules are remodelled for adult function. The resumption of fluid transport midway through the pupal stage is in preparation for a rapid diuresis at adult emergence. High rates of fluid secretion are associated with feeding or drinking in adults.     

Developmental puffing activity in the salivary gland and Malpighian tubule chromosomes ofAcricotopus lucidus (Diptera,Chironomidae)

A detailed map of the salivary gland chromosomes ofAcricotopus lucidus is presented. Differences in puffing and developmental Puffing sequences of the three salivary gland lobes were investigated from mid fourth larval instar to pupation and compared with the puffing pattern of the Malpighian tubules. The intraglandular differentiation is quite extensive; the differences in the pattern of gene activity between the anterior lobe and the main and side lobes are as great as between the salivary gland and the Malpighian tubules. In the main and side lobes all developmental puffing changes proceed synchronously whereas in the anterior lobe both asynchronous and synchronous changes occur. In the anterior lobe the asynchronous regression of BR 3 and BR 4 is followed by a characteristic sequence of activation and inactivation of puffs.     

Immune and stress response 'cross-talk' in the Drosophila Malpighian tubule

The success of insects is in large part due to their ability to survive environmental stress, including heat, cold, and dehydration. Insects are also exposed to infection, osmotic or oxidative stress, and to xenobiotics or toxins. The molecular mechanisms of stress sensing and response have been widely investigated in mammalian cell lines, and the area of stress research is now so vast to be beyond the scope of a single review article. However, the mechanisms by which stress inputs to the organism are sensed and integrated at the tissue and cellular level are less well understood. Increasingly, common molecular events between immune and other stress responses are observed in vivo; and much of this work stems of efforts in insect molecular science and physiology. We describe here the current knowledge in the area of immune and stress signalling and response at the level of the organism, tissue and cell, focussing on a key epithelial tissue in insects, the Malpighian tubule, and drawing together the known pathways that modulate responses to different stress insults. The tubules are critical for insect survival and are increasingly implicated in responses to multiple and distinct stress inputs. Importantly, as tubule function is central to survival, they are potentially key targets for insect control, which will be facilitated by increased understanding of the complexities of stress signalling in the organism.     

Mechanisms of cell volume regulation in the proximal segment of the Malpighian tubule of Rhodnius neglectus

The cell volume regulation of the lower segment cells of the Malpighian tubule of Rhodnius neglectus in anisosmotic media was evaluated by using videooptic techniques. When the medium osmolality was increased with addition of 100 mm mannitol the cells shrank to a minimum of 16.84±2.62% and subsequently swelled towards their initial volume undergoing a typical regulatory volume increase (RVI). Replacement of either K⁺ or Cl^? or HCO ₃ ^? by Na⁺, gluconate and phosphate, respectively, abolished the RVI response. Furthermore, the substitution of Na⁺ by tetramethylammonium (TMA⁺) in isosmotic conditions led to cellular swelling and death. Addition of either amiloride 10^?4 m, anthracene-9-COOH 5×10^?4 m, furosemide 5×10^?4 m or ethacrynic acid 5×10^?5 m, also abolished RVI. On the other hand, addition of either Ba²⁺ 10^?3 m, SITS 5× 10^?4 m, ouabain 10^?3 m or vanadate 10^?3 m, did not change the RVI response. When the tubules were incubated in hyperosmotic media with EGTA 2 mm or verapamil 10^?6 m, the RVI response was abolished. In contrast, a decrease of NaCl concentration from 129 to 79 mm induced a cell swelling to a maximum of 33.11+1.73%, but the cells maintained swollen, only partially regulating their volume. These results show that the proximal cells of Malpighian tubule of R. neglectus are able to regulate their volume in hyperosmotic but only partially regulating in hyposmotic solutions. The mechanisms in RVI involve Na⁺, K⁺, Cl^?, Ca²⁺ and HCO ₃ ^? transport pathways and a ouabain-insensitive ATPase stimulated by Na⁺. This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP; Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq e Financiadora de Projetos e Pesquisas-FINEP.     

Developmental changes in cell wall structure of phloem fibres of the bamboo Dendrocalamus asper

BACKGROUND AND AIMS: Bamboo culms have excellent physical and mechanical properties, which mainly depend on their fibre content and anatomical structure. One of the features which is known to contribute to the high tensile strength in bamboo is the multilayered structure of the fibre cell wall. The aim of this study was to characterize the development of the layered structure in fibre cell walls of developing and maturing culms of Dendrocalamus asper. METHODS: Cell wall development patterns were investigated in phloem fibre caps of vascular bundles in the inner culm wall areas of Dendrocalamus asper of three different age classes (<6 months old, 1 year old, 3 years old). A combination of light microscopy and image analysis techniques were employed to measure cell wall thickness and to determine number of cell wall layers, as well as to describe the layering structure of fibre walls. Two-dimensional maps showing the distribution pattern of fibres according to the number of cell wall layers were produced. KEY RESULTS: The cell walls of fibres in phloem fibre caps located in the inner part of the culm wall of D. asper developed rapidly during the first year of growth. Six different fibre types could be distinguished based upon their cell wall layering and all were already present in the young, 1-year-old culm. In the mature stage (3 years of age) the multilayering was independent of the cell wall thickness and even the thinner-walled fibres could have a large number of wall layers. The multilayered nature of cell wall structure varied considerably between individual cells and was not exclusively related to the cell wall thickness. Nevertheless, fibres at the periphery of the fibre bundles and immediately adjacent to the phloem elements exhibited a consistent and high degree of layering in their cell walls. CONCLUSIONS: The multilayered structure of fibre cell walls was formed mainly during the first year of growth by the deposition of new wall layers of variable thickness, resulting in a high degree of heterogeneity in the layering patterns amongst individual fibres. A degree of 'order' in the distribution of multilayered fibres within the caps does exist, however, with multilayered cell walls common in fibres adjacent to phloem elements and around the edge of the fibre cap. These findings confirm the observations, primarily in Phyllostachys viridi-glaucescens. The layering structure was not found to be specifically related to the thickness of the cell wall.     

Malpighian tubule polytene chromosomes of Culex quinquefasciatus (Diptera,Culicinae)

Dipteran polytene chromosomes provide an excellent model for understanding in species complexes, as well as for structural and functional cytogenetics. The status of species in the Culex pipiens complex is controversial and the use of polytene chromosomes for cytogenetic analysis in the subfamily Culicinae has been difficult because of methodological problems. In this study, Malpighian tubule polytene chromosomes were obtained from young (0 to 12 h, 20 C) and old (20 to 42 h, 28 C) laboratory-bred C. pipiens quinquefasciatus pupae. The chromosome maps for this species were constructed and compared with published data for C. pipiens pipiens and C. p. quinquefasciatus. Although the banding patterns were conserved between subspecies, analysis of the structural variations in the bands and interbands revealed differences apparently related to the physiological stage and ecogeographical strain. The organization of the centromeric regions in larval and pupal chromosomes showed greater similarity to each other than did those of pupal and adult chromosomes. The use of pupal polytene chromosomes for in situ hybridization with vector competence probes is discussed.     

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.     

Developmental changes in internal structure of chick heart plasma membrane

Although changes in electrophysiologically measurable membrane properties of chick embryo cardiac plasma membrane have been repeatedly documented during embryonic development, ultrastructural techniques were heretofore too insensitive to detect developmental changes in internal structure of this membrane. We report here significant structural changes detected by applying the quantitative analysis of Kordylewski, Karrison, and Page (Amer. J. Physiol. 245, H992-H997, 1983 and 248, H297-H304, 1985) to stereo imaged electron microscopic negatives of glutaraldehyde-fixed chick embryo hearts, freeze fractured and photographed with a goniometer stage. Between Hamburger-Hamilton stages 12+ (about 48 hr incubation) and 24 (about 96 hr incubation), plasmalemmal P-face particle density of ventricular myocytes increased from 2228 +/- 139 to 3063 +/- 109 (P less than 0.01); thereafter, measurements at stages 30, 37, 40, and 45 (7, 11, 15, and 19 days incubation) showed a slower significant linear increase which gave a least-squares line with a slope of 41 +/- 13 particles/day (P less than 0.01). Just before hatching, (stage 45) the value of 3762 +/- 234 was similar to, though slightly smaller than, the values of 4122 +/- 153 (8 days after hatching) and 4281 +/- 218 (adult chicken). These results indicate striking stage-dependent changes in the population of integral membrane proteins (channels, carriers, receptors, etc.), especially marked during early embryogenesis.     

The DNA content of polytene nuclei in midgut and Malpighian tubule cells of adultDrosophila melanogaster

Summary The amounts of DNA in midgut and Malpighian tubule cells of adult maleDrosophila melanogaster have been determined by Feulgen-DNA cytophotometry. The DNA values fall into discrete classes reflecting different levels of polyteny. The maximum level is 64C in the midgut, 256C in Malpighian tubules, and the modal values are 32C and 128C respectively. The data provide no evidence for extensive underreplication of heterochromatin. It is suggested that the reduced amount of satellite DNA found in the tissues of young adult flies may be a consequence of the fact that cycles of DNA replication started in the pre-adult stages are not completed until some hours after eclosion.     

Structure of the Malpighian tubule cells and annual changes in the structure and chemical composition of their spherites in the cave cricket Troglophilus neglectus Krauss, 1878 (Rhaphidophoridae,Saltatoria)

Periodical changes in the structure of spherites in the Malpighian tubule cells of the cave cricket Troglophilus neglectus were studied to elucidate their role during the cricket's life cycle in natural circumstances. Special interest was given to the dormant overwintering period when we hypothesized that the primary role of spherites is to supply minerals for basic vital processes. The investigation was carried out by light and transmission electron microscopy, energy dispersive X-ray spectroscopy, electron energy-loss spectroscopy and energy-filtering TEM. Spherites are present only in the middle Malpighian tubule segment, consisting of Type 1 cells, characterized, among other features, by a round, apically placed nucleus and numerous spherites, and a few Type 2 cells with an elongated nucleus in the centre and sparse spherites. At the beginning of dormancy in November juveniles, minerals are accumulated in spherites and then decline until March. In one-year-old May larvae, spherites are commonly rich in minerals, and from July onwards they are progressively exploited in the adults. Spherite destruction starts with apoptosis in senile October individuals. The findings suggest that the mineral supply of spherites in Malpighian tubules is crucial to supporting vital processes throughout the life cycle of T. neglectus.     

Developmental changes in upper airway dynamics.

Normal children have a less collapsible upper airway in response to subatmospheric pressure administration (P(NEG)) during sleep than normal adults do, and this upper airway response appears to be modulated by the central ventilatory drive. Children have a greater ventilatory drive than adults. We, therefore, hypothesized that children have increased neuromotor activation of their pharyngeal airway during sleep compared with adults. As infants have few obstructive apneas during sleep, we hypothesized that infants would have an upper airway that was resistant to collapse. We, therefore, compared the upper airway pressure-flow (V) relationship during sleep between normal infants, prepubertal children, and adults. We evaluated the upper airway response to 1). intermittent, acute P(NEG) (infants, children, and adults), and 2). hypercapnia (children and adults). We found that adults had a more collapsible upper airway during sleep than either infants or children. The children exhibited a vigorous response to both P(NEG) and hypercapnia during sleep (P < 0.01), whereas adults had no significant change. Infants had an airway that was resistant to collapse and showed a very rapid response to P(NEG). We conclude that the upper airway is resistant to collapse during sleep in infants and children. Normal children have preservation of upper airway responses to P(NEG) and hypercapnia during sleep, whereas responses are diminished in adults. Infants appear to have a different pattern of upper airway activation than older children. We speculate that the pharyngeal airway responses present in normal children are a compensatory response for a relatively narrow upper airway.     

Characterization of a leucokinin binding protein in Aedes aegypti (Diptera: Culicidae) Malpighian tubule

The insect myokinin (leucokinin-like) neuropeptide family includes peptides that have different physiological effects such as the induction of hindgut myotropic activity and stimulation of urine production. The C-terminal pentamer of myokinins Phe-X-(Ser/Pro/Ala)-Trp-Gly-amide [X=Phe, His, Asn, Ser or Tyr], had been previously determined as the minimum fragment able to elicit a functional response. The receptor(s) for these insect neuropeptides has not yet been identified. In order to characterize the Malpighian tubule leucokinin-like peptide receptor(s) from the yellow fever mosquito (Aedes aegypti), a leucokinin photoaffinity analogue (LPA) of sequence dAla-dTyr-Bpa-dLys-Phe-Phe-Ser-Trp-Gly-amide was designed based on structure/activity relationships for leucokinins. LPA caused depolarization of the transepithelial voltage (TEV) in female Malpighian tubule, confirming the activity of the peptide. The effective concentration to give half the maximum depolarization (EC₅₀) was 17 nM. The ¹²⁵I-LPA was then used to characterize leucokinin binding proteins in female Malpighian tubule membranes. It specifically labeled and saturated a protein(s) of about 54 kDa as shown by SDS-PAGE/autoradiography and by competition experiments with excess unlabeled leucokinin analogues. ¹²⁵I-LPA bound to the 54 kDa protein(s) with a K_d value of 13±3 nM in agreement with the EC₅₀ for the TEV bioassay. Altogether these data suggest that the 54 kDa protein is an Aedes-leucokinin receptor. This is the first characterization of an insect leucokinin receptor and reveals that LPA is a powerful tool to label insect myokinin receptors.     

Developmental changes in rat blood choline concentration.

1. Serum choline concentration in the newborn rat is extremely high and declines as the rat matures until adult values are attained at 20 days of age. 2. Rat milk is a rich source of choline, and rat pups denied access to milk had significantly lower serum choline concentrations than did fed littermates. We conclude that dietary intake of choline contributes to the maintenance of high serum choline concentrations in the neonatal rat. 3. In vivo, choline disappears with a half-life of 70 min. It is converted into betaine, phosphocholine and phosphatidylcholine. The rate of phosphocholine formation is identical in 3- and 10-day-old rats (3.3 mumol/h), whereas the rate of betaine formation is slower in younger animals (0.15 mumol/h at 3 days versus 0.69 mumol/h at 10 days). In vitro, choline oxidase activity [choline dehydrogenase (EC 1.1.99.1) and betaine aldehyde dehydrogenase (EC 1.2.1.8)] increased between birth and 40 days of age. The age-related acceleration in choline's conversion into betaine probably tends to diminish unesterified choline concentration in the rat.     

Ultrastructural studies on the Malpighian tubule of the pill millipede,Glomeris marginata (Villers)

Summary Ultrastructural studies on the Malpighian tubules of Glomeris marginata (Villers) reveal considerable morphological differences between the upper, fluid secreting, segment, and the lower segment which is at present of unknown function. Previous reports have shown that the upper tubule has a high permeability to compounds of high molecular weight. This may be accounted for by the fact that the epithelium shows very extensive intercellular spaces which are linked directly to junctions apparently specialised to provide a low resistance extracellular pathway between the haemocoel and the tubule lumen.Histochemical studies on the localisation of phosphatase enzymes reveal intracellular vesicles with acid phosphatase activity. The basal labyrinth of the lower tubule exhibits considerable alkaline phosphatase activity which is apparently identical in location to the enzyme revealed by two different ATPase localisation techniques.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