Biphasic perimicrovillar membrane production following feeding by previously starved Dysdercus peruvianus (Hemiptera: Pyrrhocoridae)

The development of perimicrovillar membranes (PMM) from midgut cells of starved and fed Dysdercus peruvianus was studied by using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and assays for specific enzymatic markers of the perimicrovillar membranes (alpha-glucosidase), perimicrovillar space (aminopeptidase) and microvillar membranes (beta-glucosidase). High activities of these enzymes were observed 6h post-feeding and significant production of membranes was observed at 30 h post-feeding. In the gut cells of starved insects, the rough endoplasmic reticulum was organized in concentric bundles, with a greater number of mitochondria in the cellular apex. The presence of electron dense double-membrane vesicles and the production of PMM were not observed in this condition. Thirty hours post-feeding, a disorganization of the rough endoplasmic reticulum was observed, and it was possible to see double-membrane vesicles close to the cell apex. The membrane system formation was evident with a significant development of PMM in the midgut lumen. The luminal surface of the midgut during starvation and up to 48 h post-feeding was monitored using SEM. It was demonstrated that in the starved condition, the PMM was virtually absent from gut cells, except at the base of the microvilli. At 6h post-feeding, the microvilli were already completely covered with PMM, but with a maximum of PMM formation seen at 30 h post-feeding. Signals of PMM degradation were observed 48 h after pulse feeding.     

Development and glycoprotein composition of the perimicrovillar membrane in Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae)

Hemipterans and thysanopterans (Paneoptera: Condylognatha) differ from other insects by having an intestinal perimicrovillar membrane (PMM) which extends from the base of the microvilli to the intestinal lumen. The development and composition of the PMM in hematophagous Reduviidae depend on factors related to diet. The PMM may also allow the human parasite Trypanosoma cruzi, the etiological agent of human Chagas Disease, to establish and develop in this insect vector. We studied the PMM development in the Mexican vector of Chagas Disease, Triatoma (Meccus) pallidipennis. We describe changes in the midgut epithelial cells of insects in response to starvation, and at different times (10, 15 and 20 days) after bloodfeeding. In starved insects, the midguts showed epithelial cells closely connected to each other but apparently free of PMM with some regions being periodic acid–Schiff (PAS–Schiff) positive. In contrast, the PMM was evident and fully developed in the midgut region of insects 15 days after feeding. After this time, the PMM completely covered the microvilli and reached the midgut lumen. At 15 days following feeding the labeled PAS–Schiff increased in the epithelial apex, suggesting an increase in carbohydrates. Lectins as histochemical reagents show the presence of a variety of glycoconjugates including mannose, glucose, galactosamine, N-acetyl-galactosamine. Also present were N-acetyl-glucosamine and sialic acid which contribute to the successful establishment and replication or T. cruzi in its insect vectors. By means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the formation and structure of the PMM is confirmed at 15 days post feeding. Our results confirmed the importance of the feeding processes in the formation of the PMM and showed the nature of the biochemical composition of the vectors' intestine in this important Mexican vector of Chagas disease.     

Effect of blood components, abdominal distension, and ecdysone therapy on the ultrastructural organization of posterior midgut epithelial cells and perimicrovillar membranes in Rhodnius prolixus

The effects of blood components, nerve-cord severance, and ecdysone therapy on the posterior midgut epithelial cells of 5th-instar Rhodnius prolixus nymphs 10 days after feeding were analyzed by transmission electron microscopy. Cutting the nerve-cord of the blood-fed insects partially reduced the development of microvilli and perimicrovillar membranes (PMM), and produced large vacuoles and small electrondense granules; insects fed on Ringer's saline diet exhibited well developed microvilli and low PMM production; swolled rough endoplasmatic reticulum and electrondense granules; Ringer's saline meal with ecdysone led to PMM development, glycogen particles, and several mitochondria in the cytoplasm; epithelial cells of the insects fed on Ringer's saline meal whose nerve-cord was severed showed heterogeneously distributed microvilli with reduced PMM production and a great quantity of mitochondria and glycogen in the cytoplasm; well developed microvilli and PMM were observed in nerve-cord severed insects fed on Ringer's saline meal with ecdysone; Ringer's saline diet containing hemoglobin recovered the release of PMM; and insects fed on human plasma showed slightly reduced PMM production, although the addition of ecdysone in the plasma led to a normal midgut ultrastructural organization. We suggest that the full development of microvilli and PMM in the epithelial cells depends on the abdominal distension in addition to ingestion of hemoglobin, and the release of ecdysone.     

Occurrence of midgut perimicrovillar membranes in paraneopteran insect orders with comments on their function and evolutionary significance

Hemipterans are characterized by the absence of the peritrophic membrane, an anatomical structure that envelopes the food bolus in the majority of insects. However, the microvillar membranes of many hemipteran midgut cells are not in direct contact with the food bolus, due to the existence of the so-called perimicrovillar membrane (PMM), which covers the microvilli extending into the gut lumen with dead ends. alpha-Glucosidase is a biochemical marker for PMM in the seed sucker bug Dysdercus peruvianus (Heteroptera: Pyrrhocoridae). In this article, we report that adults of the major hemipteran infra-orders (Sternorrhyncha, Auchenorrhyncha, and Heteroptera) have PMM and a major membrane bound alpha-glucosidase, which has properties similar to those of the D. peruvianus enzyme. A polyclonal antibody raised against the enzyme of D. peruvianus recognized the enzymes present in PMM from the above-mentioned hemipteran groups. The same antibody was also able of recognizing perimicrovillar alpha-glucosidase from thrips. No PMM nor membrane-bound alpha-glucosidase were found in Psocoptera and Phthiraptera midguts. This suggests that PMM and PMM-bound-alpha-glucosidase are widespread among insects of the order Hemiptera and of the sister order Thysanoptera. The data support the hypothesis that PMM may have originated in the Condylognatha (Paraneopteran taxon including Hemiptera and Thysanoptera) ancestral stock and are associated with plant sap feeding.     

Subcellular fractionation of midgut cells of the sunn pest Eurygaster integriceps (Hemiptera: Scutelleridae): Enzyme markers of microvillar and perimicrovillar membranes

The subcellular distributions of six digestive and non-digestive enzymes (α-glucosidase, β-glucosidase, alkaline phosphatase, acid phosphatase, aminopeptidase and lactate dehydrogenase) of Eurygaster integriceps have been studied. The subcellular distributions of acid phosphatase and α-glucosidase are similar and the gradient ultracentrifugation profiles of these two enzymes overlap. Two partially membrane-bound enzymes, alkaline phosphatase and β-glucosidase have similar distributions in differential centrifugation fractions, which are different from that of α-glucosidase. Sucrose gradient ultracentrifugation of membranes from luminal contents showed that β-glucosidase carrying membranes are heavier. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the profile of proteins extracted from β-glucosidase carrying membranes is different from that of α-glucosidase carrying membranes. We conclude that β-glucosidase and aminopeptidase are markers of microvillar membrane (MM) and perimicrovillar space, respectively, while α-glucosidase and acid phosphatase are perimicrovillar markers. In E. integriceps V1 luminal content is a rich source of PMM and MM and that is used to resolve these membranes.     

Alpha-Glucosidase Promotes Hemozoin Formation in a Blood-Sucking Bug: An Evolutionary History

Background

Hematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated.

Methodology/Principal Findings

Hz formation activity of an α-glucosidase was investigated. Hz formation was inhibited by specific α-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect α-glucosidase was able to inhibit Hz formation. The α-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that α-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of α-glucosidase gene, was injected into R. prolixus females'' hemocoel. Gene silencing was accomplished by reduction of both α-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of α-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of α-glucosidase shows a high similarity to the insect α-glucosidases, with critical histidine and aspartic residues conserved among the enzymes.

Conclusions/Significance

Herein the Hz formation is shown to be associated to an α-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that α-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.     

Antiserum against perimicrovillar membranes and midgut tissue reduces the development of Trypanosoma cruzi in the insect vector, Rhodnius prolixus

Antiserum raised against Rhodnius prolixus perimicrovillar membranes (PMM) and midgut tissue interfered with the midgut structural organization and reduced the development of Trypanosoma cruzi in the R. prolixus insect vector. SDS-PAGE and Western blot analyses confirmed the specific recognition of midgut proteins by the antibody. Feeding, mortality, molt, and oviposition of the insects were unaffected by feeding with the antiserum. However, the eclosion of the eggs were reduced from R. prolixus females treated with antiserum. Additionally, in vivo evaluation showed that after oral treatment with the antiserum, the intensity of infection with the Dm-28c clone of T. cruzi decreased in the digestive tract of fifth-instar nymphs and in the excretions of R. prolixus adults. These results suggest that the changes observed in the PMM organization in the posterior midgut of R. prolixus may not be important for triatomine survival but the antiserum acts as a transmission-reduction vaccine able to induce significant decreases in T. cruzi infection in the vector.     

Trypanosoma cruzi: attachment to perimicrovillar membrane glycoproteins of Rhodnius prolixus

Studies were carried out to identify proteins involved in the interface of Trypanosoma cruzi with the perimicrovillar membranes (PMM) of Rhodnius prolixus. Video microscopy experiments demonstrated high level of adhesion of T. cruzi Dm 28c epimastigotes to the surface of posterior midgut cells of non-treated R. prolixus. The parasites however were unable to attach to gut cells obtained from decapitated or azadirachtin-treated insects. The influence of carbohydrates on the adhesion to insect midgut was confirmed by inhibition of parasite attachment after midgut incubation with N-acetylgalactosamine, N-acetylmannosamine, N-acetylglucosamine, D-galactose, D-mannose or sialic acid. We observed that hydrophobic proteins in the surface of epimastigotes bind to polypeptides with 47.7, 45.5, 44, 43, 40.5, 36, 31 and 13kDa from R. prolixus PMM and that pre-incubation of lectins specifically inhibited binding to 31, 40.5, 44 and 45.5kDa proteins. We suggest that glycoproteins from PMM and hydrophobic proteins from epimastigotes are important for the adhesion of the parasite to the posterior midgut cells of the vector.     

An electron microscopic study of penetration by Trypanosoma rangeli into midgut cells of Rhodnius prolixus

The process of interaction of the Choachi strain of Trypanosoma rangeli with intestinal epithelial cells of Rhodnius prolixus was analyzed in experiments carried out in vitro and in vivo. For the in vitro experiments small fragments of the anterior region of the posterior midgut were incubated in the presence of the parasites, fixed, and processed for observation with the scanning electron microscope. Parasites attached to the surface of some epithelial cells, especially to the extracellular membrane layers (perimicrovillar membranes), were observed. For the in vivo experiments insects were infected with cultures of T. rangeli, sacrificed at different time intervals, and then processed for scanning and transmission electron microscopy. An intimate contact between the parasites and the membrane layers was observed. The parasites penetrated into cells that showed an electronlucent cytoplasm and a damaged surface, moved within the cytoplasm of the epithelial cell, reached the basal region, crossed the basal lamina, and entered the hemocoel.     

Double membrane-bounded intestinal microvilli in Oncopeltus fasciatus

A double plasma membrane (DPM) surrounding intestinal microvilli of the migratory milkweed bug, Oncopeltus fasciatus, is described. Mutant and wild types of the phytophagous insect have been studied by conventional SEM and TEM procedures with the use of membrane-enhancing staining methods. Longitudinal and transverse sections revealed a DPM surrounding microvilli and continuing over the apical portions of the intestinal cell. The outer membrane of the DPM contributes to an intestinal lining or peritrophic membrane (PTM), which apparently accumulates in layers. SEM studies reveal a rugose intestinal surface and complete PTM in both starved and fed insects. Only rarely are exposed microvilli seen by SEM. SEM examinations also enable the observation of numerous blebs on the luminal side of the PTM apparently held in position by a neck-like attachment and apparently derived from the outer membrane of the DPM. Preliminary TEM studies of microvilli revealed unique microvesicle-like structures, lying just inside the inner membrane of the DPM, which may be of membrane origin based on their typical trilaminar appearance after en bloc staining with uranyl acetate. Highly ordered microfilaments were observed to occupy the most central aspect of the microvilli.     

Purification and characterization of midgut α-amylases of Eurygaster integriceps

In the current study, midgut α-amylase from Sunn pest ( Eurygaster integriceps Puton) (Hemiptera: Scutelleridae), one of the most serious pests of wheat and barley in the wide area of the Near and Middle East, West Asia, and many of the new independent states of central Asia, were purified and characterized. Amylase activity was detected in the midgut of the insects which were collected from both over-wintering sites during winter and feeding insects during spring. Amylase activities in the midgut of over-wintering and feeding insects were 5.71 and 3.43 U/mg protein, respectively. Initially, a native electrophoretic analysis of E. integriceps crude midgut extract showed that there are two major amylase forms in the midgut. Through the sequence of ammonium sulfate precipitation, first by gel filtration chromatography (Sephadex G-75), anion exchange chromatography (diethylaminoethylcellulose) and second by gel filtration chromatography, specific activity of α-amylase of E. integriceps increased 44-fold from approximately 3 to 133 U/mg protein. Analysis of purified amylases by sodium dodecylsulfate polyacrylamide gel electrophoresis showed that these proteins had estimated molecular masses of 49 and 52 kDa. Optimum temperature was determined to be 30–40°C. The optimum pH value was 6.5 and the K _mapp for soluble starch was 0.54%.     

Effects of Changes in Feed Level, Starvation, and Level of Feed After Starvation Upon the Concentration of Rumen Protozoa in the Ovine

Four rumen fistulated sheep were used in five experiments to investigate the effect of feed level upon the concentration of rumen ciliate protozoa. The sheep were fed once daily 650 g of a pelleted diet composed of corn cobs, 45%; alfalfa meal, 35%; oats, 12.5%; cane molasses, 5%; urea, 0.4%; and vitamins and minerals, 2%. The concentration of protozoa reached minimum and maximum values at 5 and 22.5 h after feeding, respectively. Thus, to estimate apparent generation rates, concentrations of protozoa were determined at 5 and 20 h postfeeding. Apparent generation rate/h = natural log of ([concentration of protozoa at 20 h divided by concentration at 5 h] divided by the time interval, [T20 to T5]). Alteration of the feed to protozoa ratio by starvation and by changing the level of feed (200 to 900 g/day) showed that as the ratio of feed to protozoa increased, generation rate increased. Measurements of liquid turnover rates in the rumen showed that turnover rate decreased as feed level decreased. Turnover rate was near zero when the sheep were starved. Small quantities of soluble substrates, added directly to the rumen of starved sheep, maintained the protozoal population when rumen turnover was minimal. Furthermore, as rumen turnover rate increased with increased levels of feed, the effect of substrate on maintaining the protozoal population was negated. Thus, at high feed levels, turnover rate may be the dominant factor controlling the establishment and concentration of protozoa in the rumen.     

Effects of gamma irradiation on the development of Trypanosoma rangeli in the vector Rhodnius prolixus

Studies on the effects of gamma radiation on the infectivity of Trypanosoma rangeli (strain H14) for the vector Rhodnius prolixus revealed that (i) the LD(50) (lethal dose for 50% of bugs) for uninfected insects was 4147 rads; (ii) irradiated insects with a dose of 1200 rads subsequently infected with the flagellates exhibited a mortality of 45%, while uninfected irradiated insects showed a mortality of 5%, and infected nonirradiated insects exhibited 10% mortality; (iii) flagellates were present in the hemolymph of irradiated insects 7 days postinfection (p.i.), while in nonirradiated insects the parasites appeared in the hemocoel 18 days p.i.; (iv) T. rangeli infection decreased the number of hemocytes significantly and induced the formation of nodules in the hemolymph of both irradiated and nonirradiated insects; and (v) gamma irradiation affected the ultrastructural organization of the epithelial cells of the small intestine, principally the perimicrovillar membranes and microvilli. In this paper, we discuss the significance of the intestinal microenvironment of R. prolixus with regard to its interaction with T. rangeli.     

Ultrastructural and ultracytochemical study of the middle ear epithelium in the chicken,Gallus gallus domesticus

The fine structure of the epithelium lining the tympanic cavity of the chicken was studied by TEM and SEM. In addition, the distribution of nonspecific esterase activity in the epithelium was investigated by TEM. Ultrastructural study revealed the presence of disk-like apical protrusions of the epithelial cells, previously not observed in other cell types. The protrusions contained some cytoplasmic organelles and were characterized by a ring-shaped thickening around their periphery. The ring was made up of a granulo-filamentous material. Our observations clearly indicate the existence of an apocrine secretory mechanism, consisting of a progressive detachment of disk-like protrusions from the apex of the epithelial cells. The ultracytochemical study demonstrated nonspecific esterase activity on the epithelial surface and in the secretory vesicles. We propose that nonspecific esterase is a marker for middle ear surfactant in birds.     

Testosterone metabolism in the medial basal hypothalamus of the starved male rat

The conversion of testosterone to estradiol by aromatase and to dihydrotestosterone by 5 alpha-reductase was measured in the medial basal hypothalamus of starved and control male rats. Activities of both enzymes were significantly reduced in starved animals. Aromatase activity was 18.2 +/- 2.3 versus 29.8 +/- 5.7 fmol E2/mg protein/90 min (mean +/- SEM, P less than 0.02) and 5 alpha-reductase was 4.95 +/- 0.35 versus 5.96 +/- 0.30 pmol DHT/mg protein/90 min (P less than 0.02) for starved and control animals respectively. The results indicate that hypothalamic metabolism of testosterone is decreased during starvation. Therefore the increased sensitivity of the T-LH feedback described earlier in starved rats [4] cannot be explained by changes in central testosterone metabolism.     

Heme crystallization in the midgut of triatomine insects

Hemozoin (Hz) is a heme crystal produced by several blood-feeding organisms in order to detoxify free heme released upon hemoglobin (Hb) digestion. Here we show that heme crystallization also occurs in three species of triatomine insects. Ultraviolet-visible and infrared light absorption spectra of insoluble pigments isolated from the midgut of three triatomine species Triatoma infestans, Dipetalogaster maximus and Panstrongylus megistus indicated that all produce Hz. Morphological analysis of T. infestans and D. maximus midguts revealed the close association of Hz crystals to perimicrovillar membranes and also as multicrystalline assemblies, forming nearly spherical structures. Heme crystallization was promoted by isolated perimicrovillar membranes from all three species of triatomine bugs in vitro in heat-sensitive reactions. In conclusion, the data presented here indicate that Hz formation is an ancestral adaptation of Triatominae to a blood-sucking habit and that the presence of perimicrovillar membranes plays a central role in this process.     

Metabolism of amino acids in germinating yellow lupin seeds III. Breakdown of arginine in sugar-starved organs cultivated in vitro

The pathways of arginine transformations in organs of yellow lupin (Lupinus luteus L.) cultivated in vitro in the presence and absence of sucrose were investigated. Isolated embryo axes, isolated cotyledons and seeds deprived of their coat were cultured for 96 h on Heller medium with 60 mM saccharose (the fed variant, +S), without sugar (the starved variant, −S) and for 72 h without sugar, followed by 24 h in its presence (the transferred variant, −S→+S). Activities of arginine decarboxylase [EC 4.1.1.19], arginase [EC 3.5.3.1], and urease [EC 3.5.1.5] were assessed in extracts from isolated embryo axes. They were the highest in the sugar-starved variant. Supplementation of the medium with saccharose resulted in decrease in enzyme activities. The level of urea was higher (of ca. 20 %) in starved embryos than in embryos grown in the saccharose-containing medium. Moreover, participation of transamination in arginine catabolism was evidenced.     

Purification and partial characterization of a midgut membrane-bound α-glucosidase from Quesada gigas (Hemiptera: Cicadidae)

Adults of Quesada gigas (Hemiptera: Cicadidae) have a major α-glucosidase bound to the perimicrovillar membranes, which are lipoprotein membranes that surround the midgut cell microvilli in Hemiptera and Thysanoptera. Determination of the spatial distribution of α-glucosidases in Q. gigas midgut showed that this activity is not equally distributed between soluble and membrane-bound isoforms. The major membrane-bound enzyme was solubilized in the detergent Triton X-100 and purified to homogeneity by means of gel filtration on Sephacryl S-100, and ion-exchange on High Q and Mono Q columns. The purified α-glucosidase is a protein with a pH optimum of 6.0 against the synthetic substrate p-nitrophenyl α-d-glucoside and M_r of 61,000 (SDS-PAGE). Taking into account V_Max/K_M ratios, the enzyme is more active on maltose than sucrose and prefers oligomaltodextrins up to maltopentaose, with lower efficiency for longer chain maltodextrins. The Q. gigas α-glucosidase was immunolocalized in perimicrovillar membranes by using a monospecific polyclonal antibody raised against the purified enzyme from Dysdercus peruvianus. The role of this enzyme in xylem fluid digestion and its possible involvement in osmoregulation is discussed.     

Focused ion beam (FIB)/scanning electron microscopy (SEM) in tissue structural research

The focused ion beam (FIB) and scanning electron microscope (SEM) are commonly used in material sciences for imaging and analysis of materials. Over the last decade, the combined FIB/SEM system has proven to be also applicable in the life sciences. We have examined the potential of the focused ion beam/scanning electron microscope system for the investigation of biological tissues of the model organism Porcellio scaber (Crustacea: Isopoda). Tissue from digestive glands was prepared as for conventional SEM or as for transmission electron microscopy (TEM). The samples were transferred into FIB/SEM for FIB milling and an imaging operation. FIB-milled regions were secondary electron imaged, back-scattered electron imaged, or energy dispersive X-ray (EDX) analyzed. Our results demonstrated that FIB/SEM enables simultaneous investigation of sample gross morphology, cell surface characteristics, and subsurface structures. The same FIB-exposed regions were analyzed by EDX to provide basic compositional data. When samples were prepared as for TEM, the information obtained with FIB/SEM is comparable, though at limited magnification, to that obtained from TEM. A combination of imaging, micro-manipulation, and compositional analysis appears of particular interest in the investigation of epithelial tissues, which are subjected to various endogenous and exogenous conditions affecting their structure and function. The FIB/SEM is a promising tool for an overall examination of epithelial tissue under normal, stressed, or pathological conditions.