Major differences in the larval anatomy of the digestive and excretory systems of three Oestridae species revealed by micro-CT.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro-computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a 'glandular band' by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio-opaque concretions. Moreover, the anatomy of O. ovis third-instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a 'band' and anterior Malpighian tubules narrowly uniform throughout their entire length.

Transepithelial transport of P-glycoprotein substrate by the Malpighian tubules of the desert locust.

Extrusion of xenobiotics is essential for allowing animals to remove toxic substances present in their diet or generated as a biproduct of their metabolism. By transporting a wide range of potentially noxious substrates, active transporters of the ABC transporter family play an important role in xenobiotic extrusion. One such class of transporters are the multidrug resistance P-glycoprotein transporters. Here, we investigated P-glycoprotein transport in the Malpighian tubules of the desert locust (Schistocerca gregaria), a species whose diet includes plants that contain toxic secondary metabolites. To this end, we studied transporter physiology using a modified Ramsay assay in which ex vivo Malpighian tubules are incubated in different solutions containing the P-glycoprotein substrate dye rhodamine B in combination with different concentrations of the P-glycoprotein inhibitor verapamil. To determine the quantity of the P-glycoprotein substrate extruded we developed a simple and cheap method as an alternative to liquid chromatography-mass spectrometry, radiolabelled alkaloids or confocal microscopy. Our evidence shows that: (i) the Malpighian tubules contain a P-glycoprotein; (ii) tubule surface area is positively correlated with the tubule fluid secretion rate; and (iii) as the fluid secretion rate increases so too does the net extrusion of rhodamine B. We were able to quantify precisely the relationships between the fluid secretion, surface area, and net extrusion. We interpret these results in the context of the life history and foraging ecology of desert locusts. We argue that P-glycoproteins contribute to the removal of xenobiotic substances from the haemolymph, thereby enabling gregarious desert locusts to maintain toxicity through the ingestion of toxic plants without suffering the deleterious effects themselves.

The abdomen of a free-living female of Strepsiptera and the evolution of the birth organs.

Mengenillidae is a small, basal family of Strepsiptera, mainly characterized by free-living females in contrast to the endoparasitic females of Stylopidia. Here, we describe external and internal structures of the female abdomen of Eoxenos laboulbenei (Mengenillidae). The external morphology was examined and documented using microphotography. Internal structures were reconstructed three-dimensionally using a µCT-data set. The morphologically simplified abdomen comprises 10 segments. The integument is weakly sclerotized and flexible. Spiracles are present dorsolaterally on segments I-VII. Segment VII bears the posteroventral birth opening and the small abdominal segment X carries the anus at its apex. Numerous eggs float freely in the hemolymph. The musculature of segments I-IV is composed of ventral and dorsal longitudinal muscle bundles, strongly developed paramedial dorsoventral muscles and a complex meshwork of small pleural muscles, with minimal differences between the segments. Segments V-X contain more than 50 individual muscles, even though the musculature as a whole is weakly developed. Even though it is not involved in processing food, the digestive tract is well-developed. Its postabdominal section comprises a part of the midgut and the short hindgut. The midgut fills a large part of the postabdominal lumen. The lumina of the midgut and hindgut are not connected. Five or six nodular Malpighian tubules open into the digestive tract at the border region between the midgut and hindgut. The birth organ below the midgut releases the primary larvae after hatching via the birth opening at segment VII. It is likely derived from primary female genital ducts. The presence of six additional birth organs of segments I-VI are de novo formations and a groundplan apomorphy of Stylopidia, the large strepsipteran subgroup with endoparasitic females. The loss of the primary birth organ of segment VII is an apomorphy of Stylopiformia (Stylopidia excl. Corioxenidae).

Structural and ultrastructural features of the Malpighian tubules of Dolycoris baccarum (Linnaeus 1758), (Heteroptera: Pentatomidae).

The morphology and ultrastructure of the Malpighian tubules of Dolycoris baccarum were analyzed by scanning (SEM) and transmission (TEM) electron microscopy in order to determine their functional organization. The Malpighian tubules are compared with similar structures of other insects based on cell structure and functional organization. The Malpighian tubules of D. baccarum extend from the midgut-hindgut region of the digestive tract. The Malpighian tubules are divided into two regions: the proximal segment is short and flattened and the distal segment is long, stringy in shape and free in hemolymph. The tubules are generally long and narrow. There is a large number of trachea around the tubules. They consist of a single layer of epithelial cells. It is observed in the TEM observation that the epithelial cells have numerous microvilli at the apical side of the cells. At the basal side of the cells, there is a great number of membrane foldings and mitochondria among them. Besides some spherites, mitochondria, lysosome-like bodies, and large or small granules can be distinguished in the cells. With this study, we aimed to demonstrate the ultrastructure of the Malpighian tubules of D. baccarum and differences or similarities with other species.

The digestive system of the "stick bug" Cladomorphus phyllinus (Phasmida, Phasmatidae): a morphological, physiological and biochemical analysis.

This work presents a detailed morphofunctional study of the digestive system of a phasmid representative, Cladomorphus phyllinus. Cells from anterior midgut exhibit a merocrine secretion, whereas posterior midgut cells show a microapocrine secretion. A complex system of midgut tubules is observed in the posterior midgut which is probably related to the luminal alkalization of this region. Amaranth dye injection into the haemolymph and orally feeding insects with dye indicated that the anterior midgut is water-absorbing, whereas the Malpighian tubules are the main site of water secretion. Thus, a putative counter-current flux of fluid from posterior to anterior midgut may propel enzyme digestive recycling, confirmed by the low rate of enzyme excretion. The foregut and anterior midgut present an acidic pH (5.3 and 5.6, respectively), whereas the posterior midgut is highly alkaline (9.1) which may be related to the digestion of hemicelluloses. Most amylase, trypsin and chymotrypsin activities occur in the foregut and anterior midgut. Maltase is found along the midgut associated with the microvillar glycocalix, while aminopeptidase occurs in the middle and posterior midgut in membrane bound forms. Both amylase and trypsin are secreted mainly by the anterior midgut through an exocytic process as revealed by immunocytochemical data.

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.

Transcellular and paracellular pathways of transepithelial fluid secretion in Malpighian (renal) tubules of the yellow fever mosquito Aedes aegypti.

Isolated Malpighian tubules of the yellow fever mosquito secrete NaCl and KCl from the peritubular bath to the tubule lumen via active transport of Na(+) and K(+) by principal cells. Lumen-positive transepithelial voltages are the result. The counter-ion Cl(-) follows passively by electrodiffusion through the paracellular pathway. Water follows by osmosis, but specific routes for water across the epithelium are unknown. Remarkably, the transepithelial secretion of NaCl, KCl and water is driven by a H(+) V-ATPase located in the apical brush border membrane of principal cells and not the canonical Na(+), K(+) -ATPase. A hypothetical cation/H(+) exchanger moves Na(+) and K(+) from the cytoplasm to the tubule lumen. Also remarkable is the dynamic regulation of the paracellular permeability with switch-like speed which mediates in part the post-blood-meal diuresis in mosquitoes. For example, the blood meal the female mosquito takes to nourish her eggs triggers the release of kinin diuretic peptides that (i) increases the Cl(-) conductance of the paracellular pathway and (ii) assembles V(1) and V(0) complexes to activate the H(+) V-ATPase and cation/H(+) exchange close by. Thus, transcellular and paracellular pathways are both stimulated to quickly rid the mosquito of the unwanted salts and water of the blood meal. Stellate cells of the tubule appear to serve a metabolic support role, exporting the HCO(3)(-) generated during stimulated transport activity. Septate junctions define the properties of the paracellular pathway in Malpighian tubules, but the proteins responsible for the permselectivity and barrier functions of the septate junction are unknown.

Model organisms and molecular genetics for endocrinology.

Molecular endocrinology has revolutionised our understanding of neuroendocrine signalling. However, reductionist technologies emphasise the molecule at the expense of the organism. Can modern, post-genomic technologies also help us understand neuroendocrine signalling in the whole organism? The judicious use of genetic model organisms, and their associated genetic tools, can help this integrative approach. This theme is illustrated with the study of fluid balance in the fruit fly, Drosophila melanogaster. This organism is a member of the Diptera, a large Order of the largest Class of life on earth (Insecta); one with a huge impact on human health and wealth. Advanced genetic technologies allow the modulation of function of individual genes in specific cells of otherwise normal tissues in a normal animal. This organotypic context is a prerequisite for integrative physiology. Such technologies have been exploited to generate the first animals transgenic for both cytoplasmic and mitochondrial calcium reporters, and to generate a transgenic toolkit that allows cAMP, cGMP or calcium to be manipulated in any cell or tissue of choice. The results have provided new insights into neuroendocrine control in Drosophila and other organisms.

Mechanisms of elongation in embryogenesis.

Here, I discuss selected examples of elongation in embryogenesis to identify common and unique mechanisms, useful questions for further work, and new systems that offer opportunities for answering these questions. Fiber-wound, hydraulic mechanisms of elongation highlight the importance of biomechanical linkages of otherwise unrelated cellular behaviors during elongation. Little-studied examples of elongation by cell intercalation offer opportunities to study new aspects of this mode of elongation. Elongation by oriented cell division highlights the problem of mitotic spindle orientation and the maintenance of cell-packing patterns in anisotropic force environments. The balance of internal cell-adhesion and external traction forces emerges as a key issue in the formation of elongate structures from compact ones by directed migration.

In Drosophila melanogaster, the rolling pebbles isoform 6 (Rols6) is essential for proper Malpighian tubule morphology.

During myoblast fusion, cell-cell recognition along with cell migration and adhesion are essential biological processes. The factors involved in these processes include members of the immunoglobulin superfamily like Sticks and stones (Sns), Dumbfounded (Duf) and Hibris (Hbs), SH3 domain-containing adaptor molecules like Myoblast city (Mbc) and multidomain proteins like Rolling pebbles (Rols). For rolling pebbles, two differentially expressed transcripts have been defined (rols7 and rols6). However, to date, only a muscle fusion phenotype has been described and assigned to the lack of the mesoderm-specific expressed rols7 transcript. Here, we show that a loss of the second rolling pebbles transcript, rols6, which is expressed from the early bud to later embryonic stages during Malpighian tubule (MpT) development, leads to an abnormal MpT morphology that is not due to defects in cell determination or proliferation but to aberrant morphogenesis. In addition, when Myoblast city or Rac are knocked out, a similar phenotype is observed. Myoblast city and Rac are essentially involved in the development of the somatic muscles and were proposed to be interaction partners of Rols7. Because of the predicted structural similarities of the Rols7 and Rols6 proteins, we argue that genetic interaction of rols6, mbc and rac might lead to proper MpT morphology. We also propose that these interactions result in stable cell connections due to rearrangement of the cytoskeleton.

Feulgen staining in malpighian tubules of meliponid bees: a methodological contribution

Whole-mounted Malpighian tubules of larvae from two meliponid bee species fixed in acetic acid-ethanol showed apositive cytoplasmic staining with Schiff reagent when submitted to the Feulgen reaction in which acid hydrolysiswas done in 4 M HCl at room temperature. The ability of various treatments applied before the Feulgen acid hydrolysisto abolish this cytoplasmic staining was examined. The aldehyde groups of phospholipids present in the cytoplasm ofthe Malpighian tubules were blocked or removed by sequential treatment with 5% sodium borohydride and acetonechloroform(1:1, v/v) for 15 min each prior to HCl hydrolysis. This treatment is recommended in order to abolish thecytoplasmic (plasmal) reaction and to allow the reliable quantification of DNA by the Feulgen reaction and thediscrimination of nuclear phenotypes in the Malpighian tubules of meliponid bees.

Regulation of tight junction permeability with switch-like speed.

PURPOSE OF REVIEW: The case is made that tight junctions can undergo large reversible conductance changes in a matter of seconds and yet preserve their permselectivity. The diuretic peptide leucokinin transforms (renal) Malpighian tubules of the yellow fever mosquito from a moderately tight epithelium to a leaky epithelium by increasing the chloride-conductance of the paracellular shunt pathway. The nine-fold increase in the paracellular chloride-conductance brings about a non-selective stimulation of transepithelial sodium chloride and potassium chloride secretion, as expected from a conductance increase in the pathway taken by the counterion of sodium and potassium. RECENT FINDINGS: The leucokinin signaling pathway consists in part of a receptor coupled G-protein, phospholipase C, inositol-1,4,5-trisphosphate, and increased intracellular calcium concentration that bring about the increase in the paracellular, tight junction chloride-conductance. As the conductance of the tight junction pathway increases it becomes more selective for the transepithelial passage of chloride. SUMMARY: Epithelial cells in Malpighian tubules taper to tight junctions at their lateral edges exposing them directly to apical and serosal solutions. Furthermore, evolutionary pressures to excrete salt and water at high rates without the aid of glomerular filtration have led to powerful mechanisms of tubular secretion, capable of diuresis when the mosquito is challenged with the volume expansion of a blood meal. The tubular diuresis is mediated in part by increasing the paracellular chloride conductance. Thus, anatomical and physiological specializations in Malpighian tubules combine to yield the evidence for the dynamic hormonal regulation of the tight junction pathway.

Estudo morfo-fisiológico dos túbulos de Malpighi de Rhodnius prolixus stal 1859 (Hemiptera, Reduviidae, Triatominae) / Morpho-physiological study of malpighian tubules of Rhodnius prolixus stal 1859 (Hemiptera, Reduviidae, Triatominae)

Foram analisadas modificações nos túbulos de Malpighi de Rhodnius prolixus durante o ciclo alimentar do inseto. As regiões proximais e distais destes túbulos, possuem epitélio simples, envoltos por uma lâmina basal. O citoplasma é homogêneo na região proximal, existindo na distal, numerosos vacúolos, grânulos e concreções de minerais amorfos... A região proximal apresenta interdigitações basais, de aspecto vacuolizado e na região distal estas têm aspecto paralelo. O glicogênio evidenciado nos túbulos de Malpighi é possivelmente a principal fonte de reserva de energia nestas células. A partir de uma hora após a alimentação, a região proximal apresenta uma gradual diminuição do volume do epitélio, com intensa vacuolização na região basal e desorganização parcial das microvilosidades. Com 72h o volume é grande e as mitocôndrias encontram-se espalhadas pelo epitélio. A região distal apresenta concreções de minerais amorfos com aspecto de lamelas concêntricas. Após 24h observa-se muitos vacúolos de conteúdo não-eletrondenso. Os túbulos, quando submetidos a um choque hiperosmótico, apresentam uma desorganização parcial das microvilosidades na região proximal, sem alteração da vacuolização basal. Na região distal, as concreções são expelidas para o lúmen juntamente com porções de membrana, sem quebra da integridade do epitélio, mantendo o padrão em paralelo das interdigitações basais, sugerindo que os íons presentes seriam utilizados na regulação de volume. A ocorrência de uma variação na expressão da Na++K+-ATPase indica que esta enzima participa ativamente da regulação do volume. O mesmo ocorre com VH+-ATPase, cuja intensa atividade expressa com uma hora após o repasto, coincide com o pico de presença de cálcio e fósforo nas concreções de minerais amorfos, com a detecção da Ca++-ATPase por citoquímica ultraestrutural e pela alta atividade da Na++K+-ATPase neste período. O citoesqueleto evidenciou uma variação na expressão da tubulina nos diferentes tempos após a alimentação, diferente dos filamentos de actina, que mantiveram um padrão constante. Estes dados reforçam a importância da alimentação nas modificações morfológicas, na regulação do volume celular e na expressão das ATPases da membrana luminal e baso-lateral nas células dos túbulos de Malpighi de R. prolixus.

Morphology and ultrastructure of the Malpighian tubules of the Chilean common tarantula (Araneae: Theraphosidae).

Relatively little is known about the morphology and ultrastructure of the Malpighian tubules of spiders (Arachnida: Araneae). Our study represents the first investigation of the Malpighian tubules of a theraphosid spider and is the only study to examine the living Malpighian tubules using confocal laser scanning microscopy. In theraphosid spiders, the Malpighian tubules originate from the stercoral pocket in the posterior portion of the opisthosoma and extend forward toward the prosoma in a dendritic pattern. There are three distinct segments (initial, main, and terminal), all dark brown in appearance. Each segment has distinctive ultrastructural features. Both the terminal and the main segment appear to be composed of at least two cell types with finger-like cytoplasmic protrusions associated with one of these types. The terminal segment, which is most proximal to the stercoral pocket, is the largest in diameter. It is composed of large, cuboidal cells containing many mitochondria and lipid inclusions. The main segment is intermediate in diameter with many mitochondria and secretory vesicles present. The initial segment is relatively thin in comparison to the other segments and is intimately associated with the digestive gland. The cells of the initial segment contain very little cytoplasm, fewer mitochondria, secretory vesicles, and prominent inclusions.

Morphometric changes associated with sex and development in the Malpighian tubules of Aedes aegypti.

The Malpighian tubules of Aedes aegypti showed significant differences in their diameters between male and female larvae, male and female pupae, male larvae and male adults and male pupae and male adults. In every case, female values were greater than in males. Measurements of mean nuclear areas of the principal and stellate cells from Malpighian tubules, taken in males and females during development, showed that this parameter in both types of cell was significantly greater in females than in male larvae, pupae and adult stages. In males, significant differences between developmental stages were observed only in comparison with the nuclear area of larvae and adults in the principal cells, but in females, every comparison between stages showed significant differences except between pupae and adults in stellate cells. The frequency distribution of nuclear area values, in development, for male stellate and principal cells, were mostly concentrated in the first seven classes among the 30 classes considered in every stage, while for females, the frequency dropped drastically in the same classes from larvae to pupae and adults, moving to classes of higher values. Considering the importance of Malpighian tubules in insect physiology, the meaning of the differences detected are discussed on the basis of different metabolic levels, between sexes and developmental stages.

Microconcreciones de calcio en túbulos de malpinghi de rhodnius: ultraestructura y morfometría / Microconcretions of calcium in malpinghian tubules of rhodnius ultrastructure and morphometry

Los Rhodnius prolixus son insectos hematófagos que no tienen mecanismos urinarios para eliminar al exterior el exceso de calcio proveniente de la dieta. Haciendo estudios morfométricos en microfotografías de túbulos de Malpighi, hemos comparado Rhodnius con ideas bajas y altas de calcio. La concentración media de Calcio medida en las miconcreciones 21 mM (Bajo Calcio) y 1.000 mM (Alto Calcio). Estos resultados indican que hay grandes diferencias en el modelo de acumulación de Calcio entre ambos grupos. La formación de miconcreciones debe ser un mecanismo para acumular Calcio sin eliminarlo

[Patterns in the spiral formations of insects]. / Nekotorye zakonomernosti spiral'nykh obrazovanii u nasekomykh.

Regular features of spirals of chitinous filaments of tracheas were investigated in 12 species of insects. All species studied were found to have mosaic heterochirality, i.e. portions of opposite directions of curling in the trachea system, and prevalence of rightness or leftness in them. The percentage correlation of rightness-leftness are characteristic of each species and may be considered as hereditary features. The degree of prevalence of the rightness or leftness in some species as well as the degree of chiral purity (according to this parameter) is not connected with the direction of curling. The tendency to chiral purity is distinctly seen in species with both the right and left curling. As for the problem of species formation, it should be admitted that the independence of the curling direction on the one hand, and the prevalence of its degree--and the degree of chiral purity--on the other hand, speaks in favor of different mechanisms of their development. It is also confirmed by the fact that the distribution of the curling direction is referred to the whole complex of species while the degree of prevalence of any kind of curling and the degree of chiral purity are distinctive individual characteristics and are developing as a result of the adaptation selection, in connection with other features of the given species. At the same time, in accordance with some scientists it may be supposed that the directions of curling is developing on the basis of molecular structures and are thought to be accidental. Regular features of curling of trachea formations are different depending on localization of organs. For example, the tracheas feeding the malpighian vessels form spirals curling around every malpighian vessel and always to the right. Neither the trachea, nor the vessels form spirals without being in contact.

Excretion of ouabain by Malpighian tubules of Oncopeltus fasciatus.

An in vitro preparation of Malpighian tubules was used to investigate the excretion of the polar cardiac glycoside, ouabain, in Oncopeltus fasciatus. Tubules were found to consist of at least two morphologically and physiologically distinct segments, both of which metabolized ouabain. The distal segment (segment II) secreted primary urine and ouabain. Secretion of ouabain by segment II was not observed to occur against a concentration gradient and increased with increasing fluid secretion. The proximal segment (segment I) reabsorbed fluid and ouabain but not metabolites. Ouabain was reabsorbed against a strong concentration gradient (23-fold), was independent of fluid reabsorption, and increased with increasing fluid secretion by segment II. In rapidly secreting Malpighian tubules (a situation of high cardiac glycoside secretion by segment II), the presence of segment I reduced the excretion of ouabain by 84-93%, mainly by reducing ouabain concentration. It appears excretory loss of cardiac glycosides can be reduced in O fasciatus and thus may be a factor in the sequestration of cardiac glycosides in this insect.

Digestive system of poultry lice Lipeurus lawrensis tropicalis (Phthiraptera: Ischnocera).

The alimentary canal of Lipeurus lawrensis tropicalis Peters has the three typical regions but differs from that of other species of Mallophaga in some respect. The posterior region of the mid-gut is slightly broader in female than in male. The mid-gut was found to have higher epithelium not in the anterior two-thirds but in the anterior one-third and in the posterior one-third part. The epithelium is higher and convoluted in the anterior part of the hind-gut region and is greatly reduced in its posterior part. The salivary apparatus consists of a pair of salivary glands and ducts, a pair of salivary reservoirs and ducts and a salivarium. Four long blind Malpighian tubules, one dorsal, one ventral and two lateral, arise from the junction of mid- and hind-gut. Each is divisible into three distinct parts in both sexes and is without any pigmentation.