Rectal Cuticle Research Articles

Ecdysone modulates both ultrastructural arrangement of hindgut and attachment of Trypanosoma cruzi DM 28c to the rectum cuticle of Rhodnius prolixus fifth-instar nymph.

Studies on the effects of azadirachtin treatment, ecdysone supplementation and ecdysone therapy on both the ultrastructural organization of the rectum in 5th-instar nymph of Rhodnius prolixus and the ex vivo attachment behavior of Trypanosoma cruzi under these experimental conditions were carried out. Control insects had a typical and significant organization of the rectum cuticle consisted of four main layers (procuticle, inner epicuticle, outer epicuticle, and wax layer) during the entire period of the experiment. Both azadirachtin treatment and ecdysone supplementation avoid the development of both outer epicuticle and wax layer. Oral therapy with ecdysone partially reversed the altered organization and induce the development of the four main rectal cuticle layers. In the same way, the ex vivo attachment of T. cruzi to rectal cuticle was blocked by azadirachtin treatment but ecdysone therapy also partially recovered the parasite adhesion rates to almost those detected in control insects. These results point out that ecdysone may be a factor responsible - directly or indirectly - by the modulation of rectum ultrastructural arrangement providing a superficial wax layer to the attachment followed by metacyclogenesis of T. cruzi in the rectum of its invertebrate hosts.

Comportamento sexual de Anastrepha sororcula Zucchi (Diptera, Tephritidae) em laboratório

Anastrepha sororcula Zucchi, 1979, é uma das espécies de mosca-das-frutas mais disseminadas no País, sendo considerada a praga-chave que causa os maiores danos à produção de goiaba (Psidium guajava L., 1758) no Brasil. Em vista da importância desta espécie no complexo de pragas naturais da fruticultura brasileira e, em face à escassez de dados sobre sua biologia e comportamento, este trabalho teve por objetivo obter informações sobre a idade de maturação sexual de A. sororcula em laboratório e descrever seu comportamento reprodutivo. Os machos atingiram a maturidade sexual entre 7 e 18 dias após a emergência, com a maioria dos indivíduos tornando-se sexualmente maduros entre 10 e 13 dias de idade. Exibiram comportamento de sinalização às fêmeas, caracterizado pela distensão da região pleural do abdome, formando uma pequena bolsa de cada lado e, eversão de uma diminuta bolsa membranosa de cutícula retal que circunda a área anal. Durante este processo, os machos realizaram rápidos movimentos de vibração das asas, produzindo sinais audíveis. Uma gotícula foi liberada da região anal durante os movimentos de vibração alar. Após a atração das fêmeas, os machos realizaram uma série de movimentos elaborados de cortejo. As fêmeas alcançaram a maturação sexual entre 14 e 24 dias da emergência, com a maioria tornando-se sexualmente madura aos 19 dias de idade. A exibição diária das atividades sexuais foi confinada quase que exclusivamente ao período das 16:00-17:30h. A. sororcula apresentou um acentuado padrão de protandria.

Some morphological and molecular aspects of the life cycle of Trypansoma cruzi in the insect vector.

On two occasions I have reviewed the different aspects of the life cycle of Trypanosoma cruzi in the insect vector. In the first article (Zeledon 1987) I presented a detailed historical appraisal of the subject since the pioneer work done by Carlos Chagas and some years later by Emmanuel Dias in Brazil, through the more recent contributions on the subject. I also discussed some aspects of the host-parasite relationships, including the different factors involved in the cycle. In the second review article (Zeledon 1997), I stressed the microecological factors implicated in the cycle, particularly the role played by cyclic adenosine monophosphate (c AMP), a molecule which seems to be a clue in the metacyclogenesis process of the parasite. I also emphasized the importance of the morphological studies made by us and by others with both transmission and scanning electron microscopy in order to shed light on some of the key aspects of the cycle. In referring to the so called extraintestinal or hemocele cycle of the parasite in the insect we suggested, for the first time in the later review, that some of the forms described by Lacombe (1980), as part of this cycle, could belong to the same microsporidian previously described by Kramer (1972) as Octosporea carloschagasi. The description of this new species by Kramer was based on some of the forms observed by Chagas in the gut of Panstrongylus megistus which were confused by him, and 70 years later by Lacombe, with morphological stages of the life cycle of T. cruzi in the insect. In some of Chagas’ figures, Kramer recognized schizonts, sporonts and spores of the new microsporidian (see Plate 12, figs 83-85 and figs 89-97 of Chagas original paper). I now believe that the intracellular forms and the spheres described in some detail by Lacombe (1980, 1981a,b) in the hemocytes and the cells of the Malphigian tubes, represent at least part of the schizogonic and sporogonic cycles of O. carloschagasi, which were also observed in Triatoma infestans and apparently in other species of triatomines (see for instance figs 21, 22 of Lacombe 1980 and fig. 6 of Lacombe & Santos 1984). In relation to other factors involved in the cycle, some information about the population density of the different morphological stages present in various parts of the intestine of the insect, under certain physiological conditions or time periods, have been published recently (Kollien & Schaub 1997, 1998a, b). Kollien et al. (1998), also confirmed that the flagellates have no tendency to attach themselves by the flagellum to the wall of the midgut as they do in the hindgut. This group of authors also claim that the rectal cuticle of the insect consists of a superficial wax layer (hydrophobic layer) where the flagella of the parasites can attach by electrostatic forces as occurs in vitro (Kleffmann et al. 1998, Schmidt et al. 1998). This would contradict the hypothesis of a more specific type of attachment by hemidesmosomes like structures or by polypeptides as discussed previously (Zeledon 1997).

Attachment of Trypanosoma cruzi epimastigotes to hydrophobic substrates and use of this property to separate stages and promote metacyclogenesis.

In vivo, epimastigotes of Trypanosoma cruzi colonize a lipidic superficial layer of the rectal cuticle of the vector Triatoma infestans. In vitro, epimastigotes of four cultured strains and one strain from reduviids use a terminal area of the flagellum to attach to a variety of artificial hydrophobic substances, such as hydrocarbons and a range of synthetic plastics. Trypomastigotes did not attach to these substrates. Hydrophilic molecules, such as neutral or negatively charged polysaccharides, did not facilitate binding. Epimastigotes and trypomastigotes were artificially bound by electrostatic forces to positively charged chitosan or DEAE-Sephacel over their entire surface. Tween 20 and lipid-binding serum albumin effectively inhibited the hydrophobic attachment. Based on this hydrophobic interaction of epimastigotes, a new chromatography technique has been devised to gently separate trypomastigotes from epimastigotes using octacosane-coated beads. Furthermore, the in vitro transformation of epimastigotes to trypomastigotes was enhanced if epimastigotes were permitted to attach to hydrophobic, wax-coated culture vessels.

Hydrophobic attachment of Trypanosoma cruzi to a superficial layer of the rectal cuticle in the bug Triatoma infestans.

In the vector Triatoma infestans the human pathogenic flagellate Trypanosoma cruzi colonizes mainly the rectum. The rectal cuticle and associated trypanosomes were examined by electron microscopy. The cuticle of the rectum consisted of a superficial wax layer, which was not retained by conventional preparation, an outer and inner epicuticle, and a procuticle. Epimastigotes of T. cruzi attached to the superficial layer with a specialized area of the flagellum. The composition of the cuticle was analyzed by cytochemistry to determine constituents relevant for parasite attachment. Intense staining with the fluorochrome Nile red indicated the presence of lipids, and measurements of contact angles formed by test fluids with the rectal wall revealed that the luminal surface is hydrophobic. The mechanism of attachment of T. cruzi was found to be based on a hydrophobic interaction. The flagellates bound to lipids extracted from the cuticle and to saturated hydrocarbons. Chitin, which has been presumed to be the natural binding substrate of T. cruzi, was localized using gold-labeled wheat-germ lectin. Chitin occurred in the procuticle but was absent from the superficial and epicuticular layers and, thus, is not accessible for binding by T. cruzi. In addition, it could not be confirmed that galactose-specific lectins or heparin receptors mediate flagellate attachment to the rectum.

Modes of association of Trypanosoma cruzi with the intestinal tract of the vector Triatoma infestans

The interface between Trypanosoma cruzi and two regions of the intestinal tract of reduviid bugs, the small intestine and the rectum, was investigated by electron microscopy. The mode of association of the trypanosomes with the midgut surface differs fundamentally from that in the rectum, the preferred site of colonisation by T. cruzi. The parasites caused no detrimental changes in the extracellular membrane layers, microvilli or epithelial cells. Parasites resided mainly at the border of the gut contents, also regularly showing parasite–parasite interdigitations. In regions in which the extracellular membrane layers were absent or only weakly developed, trypanosome bodies or flagella occasionally could be found inserted shallowly between the tips of the microvilli. Since there were usually no ultrastructural modifications of the cell body and/or flagellum associated with attachment, there is apparently no strong attachment of the flagellates to the wall of the midgut. In the rectal lumen the flagellates also interdigitated with each other and on the rectal wall T. cruzi was intimately attached to the rectal cuticle lining. At the attachment site flagella were enlarged and sometimes contained electron-dense, hemidesmosome-like material beneath the plasma membrane.

Microspine variation in hindgut regions of four families of cockroaches (Blattaria)

Microspines in the cockroach (Blattaria) hindgut were studied to determine their structure, variation, and potential use in classification. Short spines (1–10 μm) were found in all regions of the hindgut, except for the rectal cuticle in all species, the posterior region of the ileum and colon in C. punctulatus and the posterior portion of the ileum of the blaberoid species. They were also characteristics of the pyloric and other valvular cuticle between areas of the hindgut. Moderate (20–60 μm) and long (over 100 μm) microspinal lengths were associated with bacterial aggregations or plaques in the ileum. Microspinal modifications showed only poor phylogenetic correlations.

Cellular differentiation in relation to water vapour absorption in the rectal complex of the mealworm, Tenebrio molitor

Larvae of the mealworm beetle Tenebrio molitor, are able to absorb water vapour from subsaturated air, but adults cannot. This functional difference is paralleled by structural differences in the cryptonephridial rectal complex, the site of vapour absorption in the larva. Very distinctive differences occur in the rectal pad epithelium and these are reported in detail for the first time. The cells of the larval rectal pad are very closely apposed, forming a structural, and presumably functional, unit, whereas the cells of the adult rectal pad are more clearly separate. Intracellular organization also shows clear differences. These differences indicate that the rectal epithelium may play a more important role in vapour absorption than recently ascribed to it. Other, less striking, differences in the cryptonephric Malpighian tubules and perinephric membrane as previously recorded have largely been confirmed. Morphometric analysis suggests that diffusion alone could account for the observed absorption of water vapour across the larval system from rectal lumen to the lumen of the cryptonephric tubules, but this does not rule out the possibility that other transporting mechanisms are also involved. Radial diffusion and antero-posterior gradients may be facilitated by the predominently radial and circumferential arrangement of the rectal pad cells and the surrounding cryptonephric tubules. Reinvestigation of the isolating perinephric membrane and its insertion onto the rectal cuticle supports the conclusions that insertion occurs only posteriorly. The model incorporating anterior as well as posterior insertion does not apply. The membrane posteriorly encloses a single perirectal space between rectum and tubules and in this region no perinephric or peritubular space is found between inner and outer regions of the membrane. This is the region where maximal gradients occur across the system.

Phosphate transport by locust rectum in vitro

The rectal cuticle is permeable to H2PO4−, but much less so to HPO42−. Everted rectal sacs of Schistocerca gregaria transport PO4 from lumen to hemocoel side against large concentration and electrical differences. This active process is not caused by solvent drag and it obeys Michaelis–Menten kinetics. Entry of 32PO4 into rectal tissue from the lumen is inhibited by arsenate. Much of the 32PO4 is converted to organic forms in the tissue but these do not enter the hemocoel compartment. Net rates of PO4 movement across the rectal wall in vitro are high enough to explain recovery of phosphate secreted in situ by Malpighian tubules of starved locusts. The location and possible mechanism of PO4 transport are discussed.

Rectal papillae in Musca domestica: the cuticle and lateral membranes.

The role of specialized regions of insect rectal papillae in the regulation of water and ion uptake is well documented. Although the apparatus for active uptake of water or ions is located in various cell membranes, the absorbed molecules must first pass through the cuticle which lines the rectal epithelium. Most cuticle (e.g. abdominal) has been shown to be permeable only to molecules soluble in wax, and to be impermeable to water and ions. Obviously if such cuticle lined the rectum, absorption of water and ions would be severely restricted. The present freeze-fracture and lanthanum tracer study was undertaken to investigate in more detail both the morphological features of the rectal papillae cuticle which could be responsible for its anomalous permeability and the various cell membranes involved in this transport. It has been suggested from permeability studies that the anomalous permeability of rectal papillae cuticle could be due to the lack of a complete wax layer over the surface of the rectal cuticle. The present study strongly supports this suggestion. Thus, the freeze-fracture micrographs have shown that a surface layer of the cuticle reacts during fracturing like a lipid bilayer. However, in rectal papilla cuticle this surface bilayer is interrupted at each epicuticular depression by areas of different fracturing behaviour. These discontinuities in the surface bilayer probably allow the rectal contents to contact directly the true cuticular matrix. They could, therefore, explain the case with which water and ions penetrate the rectal cuticle and so gain access to the underlying epithelial cells. Although similar discontinuities are present on some of the rectal cuticle surface external to the rectal papillae, they appear to be filled in by plugs of lipid-like material. The lateral plasma membranes of the rectal papillae cells are generally considered to be the main site of active transport. The present lanthanum tracer and freeze-fracture study has shown that the lateral plasma membranes contain 3 distinct differentiations. Septate junctions are present at the apical and basal surfaces of the epithelial layer; a further membrane differentiation is found adjacent to the septate junctions; and thirdly, an array of short, variable length, non-anastomosing linear structures covers most of the lateral plasma membrane surface. These latter structures, unlike known types of cell junctions do not show equivalent arrays in apposing membranes even when the lateral plasma membranes of adjacent cells are closely apposed. The possible function of these structures is discussed.

Compartmental Osmotic Pressures in the Rectal Complex of Tenebrio Larvae: Evidence For A Single Tubular Pumping Site

ABSTRACT The distribution of osmotic pressures derived from melting points of frozen sections of mealworm rectal complex is described. Animals rapidly frozen during atmospheric absorption show radially increasing values from lumen to Malpighian tubules suggesting that water uptake is driven by a single pump located in the area of highest solute concentration. Volume–concentration relationships of isolated rectal cuticle and perirectal fluid are consistent with their transmitting water passively to an area of lower solvent activity. The rectum of animals frozen with some delay after atmospheric absorption show high osmotic pressures with no radial gradients, a condition which is thought to exist when the rectum is in its faecal dehydration mode. Rectal osmotic pressures in all absorbing animals increased towards the posterior end. Several, presumably extreme posterior tubular, osmotic pressures agree well with the 6·7 osmol.kg−1 value expected for an 88% R.H. threshold for atmospheric absorption.

Symmetry and non-linearity of osmotic flow across rectal cuticle of the desert locust.

ABSTRACT A method for measuring osmotic flow of water across rectal cuticle of the desert locust in vitro is described. The osmotic permeability coefficient decreases as the average osmolality of the two bathing solutions increases, probably due to dehydration of the cuticle. This is supported by a corresponding decrease in the diffusion permeability coefficient, as measured by flux of tritiated water in the absence of osmotic flow. Osmotic permeability is at least fifty times greater than diffusion permeability. This supports the hypothesis that osmotic flow occurs by bulk flow or single-file diffusion through pores. Isotopic flux measurements during net flow support this view. The osmotic permeability of the cuticle differs by less than 25% if at all, in the two directions of flow; that is, rectification or ‘valve-like’ behaviour is not significant. The cuticle represents only 2·6 to 13% of the total resistance to osmotic flow across the whole rectal wall and is not responsible for the asymmetry of the latter. Application of Beament’s model for water transport to the rectal wall of insects can be excluded in the case of the desert locust.

Molecular sieving of hydrophilic molecules by the rectal intima of the desert locust (Schistocerca gregaria).

ABSTRACT The permeability of perfused intimal sacs to fourteen non-ionic, hydrophilic molecules of graded molecular size was estimated by radioisotope flux. The rectal cuticle acts as a molecular sieve severely restricting the rate of penetration of molecules with increasing hydrated size. The penetration of test molecules was as predicted by the Renkin equation for a uniform population of water-filled pores having radii of 6.5 Å. The properties of cuticles from the rectum and the integument are compared and the role of the rectal intima in the excretory process in the desert locust is discussed.