Biochemical changes in the transition from vitellogenesis to follicular atresia in the hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae).

In this work, we have explored the biochemical changes characterizing the transition from vitellogenesis to follicular atresia, employing the hematophagous insect vector Dipetalogaster maxima as a model. Standardized insect rearing conditions were established to induce a gradual follicular degeneration stage by depriving females of blood meal during post-vitellogenesis. For the studies, hemolymph and ovaries were sampled at representative days of pre-vitellogenesis, vitellogenesis and early and late follicular atresia. When examined by scanning electron microscopy, ovarioles at the initial stage of atresia were small but still showed some degree of asynchronism, a feature that was lost in an advanced degeneration state. At late follicular atresia, in vivo uptake assays of fluorescently labeled vitellogenin (Vg-FITC) showed loss of competitiveness of oocytes to uptake vitellogenin. Circulating vitellogenin levels in atresia were significantly higher than those registered at pre-vitellogenesis, most likely to maintain appropriate conditions for another gonotrophic cycle if a second blood meal is available. Follicular atresia was also characterized by partial proteolysis of vitellin, which was evidenced in ovarian homogenates by western blot. When the activity of ovarian peptidases upon hemoglobin (a non-specific substrate) was tested, higher activities were detected at early and late atresia whereas the lowest activity was found at vitellogenesis. The activity upon hemoglobin was significantly inhibited by pepstatin A (an aspartic peptidase inhibitor), and was not affected by E64 (a cysteine peptidase inhibitor) at any tested conditions. The use of specific fluorogenic substrates demonstrated that ovarian homogenates at early follicular atresia displayed high cathepsin D-like activity, whereas no activity of either, cathepsin B or L was detected. Mass spectrometry analysis of the digestion products of the substrate Abz-AIAFFSRQ-EDDnp further confirmed the presence of a cathepsin D-like peptidase in ovarian tissue. In the context of our findings, the early activation of cathepsin D-like peptidase could be relevant in promoting yolk protein recycling and/or enhancing follicle removal.

Oogenesis and egg development in triatomines: a biochemical approach

Em triatomíneos, assim como em outros insetos, o acúmulo de vitelo é um processo no qual um tecido extraovariano, o corpo gorduroso, produz proteínas que são empacotadas no interior de um ovo. A principal proteína, sintetizada pelo corpo gorduroso, que é acumulada no interior de um ovócito, é a vitelogenina. Este processo é também conhecido por vitelogênese. Existem crescentes evidências em triatomíneos, que além do corpo gorduroso, o ovário também produz proteínas de vitelo. A forma como estas proteínas de vitelo entram nos ovócitos será aqui comentada. O vitelo é um material complexo composto por proteínas, lipídeos, carboidratos e outros compostos minoritários que são empacotados de uma maneira organizada no interior dos ovócitos. A fertilização dispara a embriogênese, um processo que culmina com o desenvolvimento do embrião. Durante a embriogênese o vitelo será utilizado para a construção de um novo indivíduo, a ninfa de primeiro estádio. O desafio para a próxima década é entender onde e como estas proteínas de vitelo são utilizadas junto com os seus componentes não protéicos, em compasso com o programa genético do embrião, que comanda a diferenciação celular (fase inicial da embriogênese) e diferenciação do embrião (fase final da embriogênese) no interior do ovo.

Role of salivary antihemostatic components in blood feeding by triatomine bugs (Heteroptera).

Salivary gland homogenates from 4 genera of triatomine bugs were assayed for anticlotting, apyrase, and vasodilatory activities, and these activities were correlated with the efficiency of each bug species to initiate a blood meal. Antihemostatic activities spanned a large range of values. Apyrase activity in members of the genus Rhodnius was markedly different from that in other genera with respect to their sensitivity to divalent cation activators. Apyrase and vasodilatory activities, but not anticlotting activity, correlated with feeding efficiency of bugs taking a blood meal on a rat. Results are discussed within the context of the evolution of blood-feeding by insects.

Metabolic post-feeding changes in flat body and hemolymph of Dipetalogaster maximus (Hemiptera: Reduviidae)

Lipids and glycogen in fat body as well as the modifications in the wet weight of this organ were evaluated in an unfed insect, Dipetalogaster maximus, on day 5 after adult acdysis (time 0) and during a 30-day period after ingestion of blood meal. Total lipids, high density lipophorin (HDLp), carbohydrates, total protein and uric acid were determined in the hemolymph during the same period. Fat body wet weight was maximum on day 10 post-feeding and represented on day 30 only 42 per cent of the maximum weight. Lipids stored in the fat body increased up to day 15 reaching 24 per cent of the total weight of tissue. Glycogen was maximum on day 20, representing approximately 3 per cent of the fat body weight. HDLp represented at all times between 17-24 per cent of the total proteins, whose levels ranged between 35 and 47 mg/ml. Uric acid showed at 20, 25 and 30 days similar levels and significantly higher than the ones shown at days 10 an 15. Hemolymphatic lipids fluctuated during starvation between 3-4.4 mg/ml and carbohydrates showed a maximum on day 15 after a blood meal, decreasing up to 0.26 mg/ml on day 25. The above results suggest that during physiological events such as starvation, the availability of nutrients is affected, involving principally the fat body reserves.

Preservaçäo do Trypanosoma cruzi em tritomíneos alimentos em galinha e em camundongo / Trypanosoma cruzi preservation in triatomines fed in chicken and mouse

Estudou-se a preservaçäo do Trypanosoma cruzi em diferentes espécies de triatomíneos, alimentados em galinha e em camundongo: Rhodnius prolixus (Stal, 1859), R. robustus (Larrouse, 1927), Triatoma brasiliensis (Neiva, 1911) e T. rubrovaria (Blanchard, 1843). Os triatomíneos infectaram-se com o T. cruzi, em camundongos com infecçäo aguda. Para estimar a permanência do T. cruzi nos triatomíneos examinaram-se, individualmente, 20 insetos de cada espécie. Os triatomíneos permaneceram infectados pelo T. cruzi, tanto em camundongo quanto em galinha, ao longo de 5 meses de observaçäo, sem definir uma alteraçäo significativa da influência da fonte sangüinea sobre a replicaçäo do tripanosoma

An inhibitor of collagen-induced platelet aggregation from the saliva of Triatoma pallidipennis.

The saliva of Triatoma pallidipennis, a blood-sucking triatomine bug (Hemiptera, family Reduviidae, subfamily Triatominae) was found to contain a factor that specifically inhibits collagen-induced platelet aggregation. The 19-kDa protein was purified to homogeneity and named pallidipin. Collagen-mediated aggregation of platelets in plasma and of washed platelets was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effectors (ADP, thrombin, thromboxane A2 mimetic U46619, phorbol ester) was detected. Pallidipin had no effect on platelet adhesion to collagen but inhibited ATP release from platelets. It interacted reversibly with platelets and may share with collagen a common target on them. The protein exhibits a unique primary structure (predicted from cDNA clones) with no significant similarity to other previously described sequences. The protein produced in recombinant baby hamster kidney cells had antiaggregatory effects similar to those of native pallidipin. Availability of recombinant pallidipin will allow further investigation of the precise mechanism of action.

Cyclic AMP in the Malpighian tubule fluid and in the urine of Rhodnius prolixus.

We demonstrate the presence of cyclic adenosine monophosphate (cAMP) in the fluid secreted by isolated Malpighian tubules of Rhodnius prolixus. In addition, we show that fifth-instar R. prolixus excrete cAMP in the urine after a meal of human blood. Nonstimulated isolated Malpighian tubules secrete small amounts of cAMP that increase about 10-fold after the addition of 5-hydroxytryptamine (5-HT). 5-HT is known to mimic R. prolixus diuretic hormone. The present findings demonstrate that 5-HT also acts via cAMP. The presence of cAMP in the rectal sac of the insect could be of importance in the differentiation of Trypanosoma cruzi and in the cycle of Chagas disease.

[Biochemistry of the development cycle of Triatoma infestans (vinchuca). X. Hemolymph lipoproteins of females]. / Bioquímica del ciclo evolutivo del Triatoma infestans (vinchuca). X. Lipoproteinas hemolinfaticas de hembras.

A lipoprotein of high density (HDL) and three of very high density (VHDL-I, VHDL-f and VHDL-II) were separated by ultracentrifugation from T. infestans female hemolymph. Lipids were mainly transported by HDL, whereas VHDL-II presented the highest protein content. In all the lipoproteins, 1,2 and 1,3-diacylglycerols, triacylglycerols, free fatty acids, cholesterol, cholesterol esters and hydrocarbons, were present. Phosphatidylcholine and phosphatidylethanolamine were also identified. The main lipids were represented by phospholipids, 1,2 and 1,3-diacylglycerols and hydrocarbons. All lipoproteins were delipidated to study the corresponding apolipoproteins. They were examined by SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Apo-HDL was separated into four intense polypeptide bands of approximate MW 19,000; 45,000; 86,000 and 200,000. Apo-VHDL-I was fractionated into three polypeptide bands--a major one of MW 18,500 and two minor ones of 28,000 and 45,000. The apolipoproteins belonging to VHDL-f that only appeared in females, were separated into 10 bands; the major ones corresponded to MW of 58,000, 86,000 and 160,000. In these apolipoproteins, the specimens of 58,000 and 160,000 showed positive carbohydrate reaction. Yet, in the apo-VHDL-II the most intense protein subunits primarily corresponded to bands of MW 160,000 and 86,000. Apparently, the four lipoproteins would share two polypeptide chains.

Chitin synthesis in Triatoma infestans and other insects.

In vivo and in vitro synthesis of chitin in Triatoma infestans was studied. For in vivo experiments, [14C] sugars were injected through the abdominal wall. Maximal incorporation of radioactivity into the cuticle was attained immediately after the ecdysis. The identification of in vivo synthesized chitin was performed by the enzymatic hydrolysis of the alkali-insoluble material from the cuticle with Helix chitinase. The main water-soluble compound obtained was N-acetylglucosamine as demonstrated by chromatographic procedures. In vitro synthesis of chitin was carried out with enzymatic crude extracts from Triatoma infestans, and UDP-N-acetylglucosamine was used as "source" of glycosyl moieties. Higher amounts of [14C] N-acetylglucosamine incorporation to chitin than those previously reported by others, were obtained. The identity of the product was confirmed in a similar way as that from in vivo synthesis. Radioactivity was also found in a liposoluble fraction concomitantly with chitin synthesis. This compound had an anionic behavior, was acid labile and had similar chromatographic properties as dolichol pyrophosphate N-acetylglucosamine obtained with pig liver extracts. Knowledge about dolichol phosphate sugars mediated glycoprotein synthesis in eukaryotes, suggests the involvement of this type of N-acetylglucosaminyl-phospholipid in macromolecule "building" even in insects.

Incorporation of lipid into the epicuticle of Rhodnius (Hemiptera).

The incorporation of lipid into both the outer and inner epicuticle during deposition is described. Waterproofing of the epicuticle by secretion of the wax layer, and sclerotization with or without melanization, are controlled from a distance by the epidermal cells by way of the pore canals. The pore canals gradually narrow as they approach the epicuticle. On reaching the inner epicuticle the canal ends in a conical projection from the apex of which a permeable lipophilic channel about 20-25 nm in diameter runs vertically to the apex of which a permeable lipophilic channel about 20-25 nm in diameter runs vertically to the surface. Shortly before ecdysis, silver-binding material (perhaps protein rich in tyrosine, or other precursors concerned in sclerotization) spreads radially from a point in the cuticular channels just below the outer epicuticle, and gradually impregnates the outer two thirds or more of the inner epicuticle. The precise pattern varies in different cuticular structures. Argentaffin materials (polyphenols) first appear in these same sites at the time of ecdysis and increase rapidly during the next 24 h. Lipid appears in the lumen of the distal parts of the pore canals (with a patchy distribution) shortly before ecdysis. When digestion and absorption of the old endocuticle are almost complete, minute lipid droplets appear on the surface of the epicuticle, apparently exuded from the epicuticular channels, and spread to make a uniform layer. When first formed this layer strains readily with Sudan B, but the lipid becomes incorporated in a delicate non-lipid silver-binding membrane (also exuded from the epicuticular channels) and hardens just before ecdysis, to form the so-called 'wax layer' which then no longer stains with Sudan B. Within half an hour after ecdysis the alcian blue-staining cement layer is poured out by the dermal glands, and forms a continuous but somewhat irregular covering over the 'wax layer'. Changes in the epicticle that accompany the repair of abrasions are described.

E a fisiología de los triatominos con especial referencia a la alimentación por sangre / The physiology triatominae with special reference to feeding by blood

Se revisa literatura existente sobre alimentación, procesos post-alimenticios y vuelvo de triatominos (Rhdnius prolixus, Triatoma infestans y Panstrongylus megistus fueron las especies más estudiadas), considerándose aspectos de la conducta alimentaria, localización de huéspedes, digestión, nutrición, metabolismo, excreción, requerimientos nutricionales, comensalismo (simbiontes), efectos de la carencia de alimentos, papel de la alimentación en el desarrollo, ecdisis y reproducción; y aspectos del vuelo: iniciación, balancehídrico durante el mismo y metabolismo (mencionándose el papel de los aminoácidos como fuente energética)

Aspectos del metabolismo lipídico en triatominos / Aspectos of lipid metabolism in triatominae

Se estudian aspectos del metablismo de lípidos (considerados como fuente de energía) en triatominos (enfatizándose en Triatoma infestans)-que reciben en la sangre ingerida los lípidos que metabolizarán y utilizarán para sus funciones-; analizándose el contenido de lípidos en el crecimiento; la biosíntesis de lípidos y ácidos grasos, el transporte de lípidos por hemolinfa y; la composición lipídica de la hemolinfa, la epicútúcula, los ácidos grasos, las lipoproteínas y los ácidos volátiles de insectos

Avances en la línea entomológica y control integrado / Advances in the line entomologycal and integrated control

Discute los resultados obtenidos en estudios sobre triatominos, realizados en la línea entomológica y de control integrado, del Progrma Nacional de Investigación en Enfermedades Endémicas (Argentina), dentro de las áreas de: bioquímica, toxicología y control de triatominos (metabolismo de lípidos y de hormonas, caracterización de vitelogeninas, toxicodinámica de insecticidas, y, metodologías de control químico); morfología, sistemática, biología y ecología de triatominos y sus enemigos naturales); morfología de diversas estructuras cuticulares vinculadas con órganos sensoriales y estridulatorios, composición de substancias volátiles liberadas por glándulas epidérmicas; y, aspectos del comportamiento de insectos