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Alimentação do Rhodnius prolixus, no laboratório
... A alimentação artificial supre as necessidades nutricionais desses insetos, sem os inconvenientes da manutenção de hospedeiros vivos, além de minimizar efeitos de características individuais dos hospedeiros nos estudos de transmissão do Trypanosoma cruzi. Este método foi também utilizado como isca em medidas de controle, conforme demonstrado por Lima et al. (1991Lima et al. ( , 1992 e Lima (1994); para manutenção de colônias em laboratório (Garcia et al. 1975), além da utilização no xenodiagnóstico artificial. Neste último caso torna-se ainda mais indicado, já que podem ocorrer casos de reações alérgicas cutâneas às picadas desses insetos (Mott et al. 1980, Costa et al. 1981. ...
... Ingestão de sangue -Observou-se que o 2º repastos, quando alimentado em pombos, para alcançar a fase seguinte, média essa superior à obtida no presente trabalho em alimentação artificial, que foi de um repasto. A regularidade nos períodos de intermuda registrada no presente trabalho foi observada também por Garcia et al. (1975) para R. prolixus Stal, 1859 e por Lent e Valderrama (1977) para R. pictipes. ...
... O número de ovos produzidos está diretamente relacionado à quantidade de sangue ingerido (Buxton 1930, Friend et al. 1965, Regis 1979. Garcia et al. (1975) a: Friend et al. 1975;b: Buxton, 1930;c: Brasileiro & Perondini, 1974;d: Zeledón et al. 1970 a e b;e: Rabinovich, 1972;f: Wood, 1976 2º 8,7 mg; 3º 13,5 mg; 4º 17,8 mg; 5º124 mg; machos 67,5mg e fêmeas 100,5mg. Esses resultados são inferiores aos obtidos no presente trabalho, para ambos os grupos, exceto para o 5º estádio. ...
Rhodnius pictipes (Hemiptera, Reduviidae) from Serra Norte, State of Pará, Brazil, acclimatized in an insectary at the Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Departamento de Entomologia, Instituto Oswaldo Cruz, were fed through a silicone membrane. In order to know the viability and the efficiency of this membrane compared with insects fed on mice, the number of bloodmeals taken, period of development of the five nymphal instars, longevity of adults, average amount of blood intake in each meal and percent of mortality were observed. A total of 310 insects, were used, comprising 50 nymphs of each instar, as well as 30 male and 30 female adults. Insects fed artificially had reduced minimal and maximal periods of development than the group fed on mice. The largest relative increase of body weight was observed in the 2nd instar followed by the 1st, and the amount of blood ingested increased during the development, to the 5th instar for both groups. There were no significant differences between the groups fed artificially and in vivo according to Tukey's test for p > 0.05. The percent of mortality in the 1st instar was 18% for artificially fed and 16% for the group fed on mice; these percentages decreased as insects developed until the 4th instar, without mortality, returning to increase in the 5th instar. R. pictipes was shown to be easily adaptable to artificial feeding, and could be considered as an important and viable experimental model.
... The insects used were normal mated females having their second blood meal as an adult. Experimental animals were fed with rabbit blood using an artificial feeding apparatus (Garcia et al., 1975). ...
... To investigate the effect of different levels of heme-RHBP on the number of eggs produced, the insects were fed with the same volume of rabbit blood, plasma, and plasma enriched with 1 mM hemin in an artificial feeding apparatus (Garcia et al., 1975). (During 16 days after this meal, the eggs were collected, and counted daily for 16 days). ...
The uptake of RHBP (Rhodnius heme-binding protein) by the ovaries of Rhodnius prolixus was characterized. RHBP purified from oocyte was labeled with 125I and used to study the process of uptake by the ovary in vivo and in vitro. After injection, the [125I]RHBP was readily removed from the hemolymph and accumulated especially in the ovary. The capacity of the ovary to take up [125I]RHBP from the hemolymph varied during the days following blood meal. It increased up to day 2, remained stable until day 5, and then decreased up to the end of oogenesis. In vitro, the uptake of [125I]RHBP was linear at least up to 60 min. The uptake was dependent on [125I]RHBP concentration and showed to be a saturable process. The addition of a molar excess of non-related proteins such as Vitellin (Vt), Lipophorin (Lp), and Bovine Serum Albumin (BSA) did not reduce [125I]RHBP uptake. Using immunogold technique the RHBP was localized at the microvilli, coated pits, and yolk granules. The main yolk protein, Vt, did not compete with RHBP for the uptake. Thus, it is discussed here that they bind to independent binding sites of the oocytes, and are directed later on to the same compartment. The need of both proteins for the completion of mature oocyte was verified in vivo. The reduction of heme-RHBP in the hemolymph, by changing the diet, decreased the number of eggs laid. Increasing the concentration of heme-RHBP in the hemolymph, the number of eggs produced increased in a dose dependent manner. In vitro, both apo-RHBP and heme-RHBP can be taken up by the oocyte. Since the mature oocyte contains only heme-saturated RHBP, the possible fate of apo-RHBP is also discussed. Arch. Insect Biochem. Physiol. 39:133–143, 1998. © 1998 Wiley-Liss, Inc.
... The insects used were normal mated females having their second blood meal as an adult. Experimental animals were fed with rabbit blood using an artificial feeding apparatus (Garcia et al., 1975). ...
... To investigate the effect of different levels of heme-RHBP on the number of eggs produced, the insects were fed with the same volume of rabbit blood, plasma, and plasma enriched with 1 mM hemin in an artificial feeding apparatus (Garcia et al., 1975). (During 16 days after this meal, the eggs were collected, and counted daily for 16 days). ...
The uptake of RHBP (Rhodnius heme-binding protein) by the ovaries of Rhodnius prolixus was characterized. RHBP purified from occyte was labeled with 125I and used to study the process of uptake by the ovary in vivo and in vitro. After injection, the [125I]RHBP was readily removed from the hemolymph and accumulated especially in the ovary. The capacity of the ovary to take up [125I]RHBP from the hemolymph varied during the days following blood meal. It increased up to day 2, remained stable until day 5, and then decreased up to the end of oogenesis. In vitro, the uptake of [125I]RHBP was linear at least up to 60 min. The uptake was dependent on [125I]RHBP concentration and showed to be a saturable process. The addition of a molar excess of non-related proteins such as Vitellin (Vt), Lipophorin (Lp), and Bovine Serum Albumin (BSA) did not reduce [125I]RHBP uptake. Using immunogold technique the RHBP was localized at the microvilli, coated pits, and yolk granules. The main yolk protein, Vt, did not compete with RHBP for the uptake. Thus, it is discussed here that they bind to independent binding sites of the oocytes, and are directed later on to the same compartment. The need of both proteins for the completion of mature oocyte was verified in vivo. The reduction of heme-RHBP in the hemolymph, by changing the diet, decreased the number of eggs laid. Increasing the concentration of heme-RHBP in the hemolymph, the number of eggs produced increased in a dose dependent manner. In vitro, both apo-RHBP and heme-RHBP can be taken up by the oocyte. Since the mature oocyte contains only heme-saturated RHBP, the possible fate of apo-RHBP is also discussed.
... Our bed bugs are fed defibrinated rabbit blood (purchased from Hemostat Laboratories: Dixon, CA) once per week through the use of an artificial feeding system ( Figure A-4).This system, originally developed by Garcia et al. (1975), and later modified by Montes et al. (2002), effectively imitates the elevated body temperature and epidermis of a bed bug host. The system functions by using a hot water bath (Isotemp Fisher Scientific Model 2239) coupled with a water pump (GeoGlobal Partners Model FP80: ...
... Adult females of R. prolixus were fed in an artificial feeder (Garcia et al., 1975) on whole rabbit blood supplemented with 3 H-cholesterol, 10 6 disintegrations per minute (DPM)/ml of blood ([1,2-3 H(N)]-cholesterol, specific activity 51 Ci/mmol; PerkinElmer, Boston, MA, United States; 3 ng of 3 H-cholesterol/ml of blood). About 1 h after blood meal, insects were individually housed at 28 • C. On the first, third, fifth, seventh, and 10th days after the 3 H-cholesterol-enriched blood meal, hemolymph was individually collected in the presence of phenylthiourea. ...
Insects are unable to synthesize cholesterol and depend on the presence of sterols in the diet for cell membrane composition and hormone production. Thus, cholesterol absorption, transport, and metabolism are potential targets for vector and pest control strategies. Here, we investigate the dietary cholesterol absorption and tissue distribution in the kissing bug Rhodnius prolixus using radiolabeled cholesterol. Both the anterior and posterior midguts absorbed cholesterol from the ingested blood, although the anterior midgut absorbed more. We also observed esterified cholesterol labeling in the epithelium, indicating that midgut cells can metabolize and store cholesterol. Only a small amount of labeled cholesterol was found in the hemolymph, where it was mainly in the free form and associated with lipophorin (Lp). The fat body transiently accumulated cholesterol, showing a labeled cholesterol peak on the fifth day after the blood meal. The ovaries also incorporated cholesterol, but cumulatively. The insects did not absorb almost half of the ingested labeled cholesterol, and radioactivity was present in the feces. After injection of ³H-cholesterol-labeled Lp into females, a half-life of 5.5 ± 0.7 h in the hemolymph was determined. Both the fat body and ovaries incorporated Lp-associated cholesterol, which was inhibited at low temperature, indicating the participation of active cholesterol transport. These results help describe an unexplored part of R. prolixus lipid metabolism.
... Adults females of R. prolixus were maintained at 28°C and 80-90 % relative humidity in the R. prolixus Colony at the Institute of Medical Biochemistry at the Federal University of Rio de Janeiro, Brazil. The insects were fed upon a membrane apparatus containing citrated blood (Garcia et al., 1975). All animal care and experimental protocols were conducted following the guidelines of the institutional animal care and use committee (Committee for Evaluation of Animal Use for Research from the Federal University of Rio de Janeiro, CAUAP-UFRJ) and the NIH Guide for the Care and Use of Laboratory Animals (ISBN 0−309-05377−3). ...
Rhodnius prolixus is an important vector of Trypanosoma cruzi, the etiological agent of Chagas disease. Insect adults have a pair of Metasternal Glands (MGs) and the secretion emitted by these glands acts as sex pheromone. Recent studies have focused on the chemical composition of this pheromone, electrophysiological responses to MGs compounds and mating behavior assays. Morphological studies of these glands are still scarce. Thus, considering the relevance of MGs in the sex pheromone biosynthesis, we investigated the morphology and ultrastructure of R. prolixus MGs. The glandular apparatus presents a tubular structure containing secretory cells with canalicules that fuse with the central duct which conducts the secretion to a pear-shaped reservoir connected to the exterior by a droplet-shape orifice. The secretory cells are classified as class III, they present a well-developed rough and smooth endoplasmic reticulum. Smooth endoplasmic reticulum is a site of lipid biosynthesis that may be involved in the mevalonate pathway, a probable route of the sex pheromone biosynthesis in this insect. The presence of rough endoplasmic reticulum indicates a possible peptides/proteins secretions site which were still not characterized in MGs. Several mitochondria are scattered in the cytoplasm that may suggest a high metabolic activity. Further studies should be carried out to correlate these data with the sex pheromone biosynthesis in this vector.
... Nymphs of 4th and 5th instars of Triatoma infestans, kindly provided by the Triatomine Collection of Fiocruz, were employed to assess the ability of new bat trypanosomes for infecting triatomines. Infection was assessed by artificial xenodiagnoses method using rabbit erythrocytes mixed (v/v) with bat trypanosome cultures incubated at 37°C to feed the triatomines according Garcia et al. (1975). About forty triatomines were fed with each culture, and at 15, 30, and 45 days post-feeding ten triatomines were dissected, and the presence of trypanosomes in the digestive tube, hemolymph and salivary glands were microscopically investigated at each time point. ...
Molecular phylogenetic studies have revealed the growing diversity of bat trypanosomes. Here, 14 isolates from blood samples of the vampire bat Desmodus rotundus (Phyllostomidae) from Rio de Janeiro, Southeast Brazil, were cultivated, and morphologically and molecularly characterized. All isolates represent a novel species named Trypanosoma madeirae n. sp. positioned in the Neobat lineage of the clade T. cruzi. The Neobat lineage also comprises closely related trypanosomes of clades Neotropic 1, 2 and 3 from diverse phyllostomid species. Trypanosomes of Neotropic 1, found in Trachops cirrhosus and Artibeus jamaicensis (phyllostomids), likely represent a different species or genotype closely related to T. madeirae. Consistent with its phylogenetic positioning, T. madeirae differs from Trypanosoma cruzi in morphology of both epimastigote and trypomastigote culture forms and does not infect Triatoma infestans. Similar to its closest relatives of Neobat lineage, T. madeirae was unable to develop within mammalian cells. To date, PCR-surveys on archived blood/liver samples unveiled T. madeirae exclusively in D. rotundus from Southern to Northern Brazil. The description of a new species of bat trypanosome associated with vampire bats increases the repertoire of trypanosomes infecting D. rotundus, currently comprised of Trypanosoma cruzi, T. cruzi marinkellei, Trypanosoma dionisii, Trypanosoma rangeli, Trypanosoma pessoai, and Trypanosoma madeirae.
... Bed bugs were not selected by sex so that an accurate representation of the population could be obtained. All vials were fitted with mesh tops secured with rubber bands to allow bed bugs to feed through the mesh on an artificial feeding system (developed by Garcia et al. [27] and modified by Montes et al. [28]). All bed bugs were allowed to feed to repletion on defribinated rabbit blood (Hemostat Laboratories, Dixon, CA) by exposing vials containing bed bugs to the artificial feeding system for~30 minutes. ...
We observed that teneral adults (<1 h post-molt) of Cimex lectularius L. appeared more adept at climbing a smooth surface compared to sclerotized adults. Differences in climbing ability on a smooth surface based on sclerotization status were quantified by measuring the height to which bed bugs climbed when confined within a glass vial. The average maximum height climbed by teneral (T) bed bugs (n = 30, height climbed = 4.69 cm) differed significantly (P< 0.01) from recently sclerotized (RS) bed bugs (n = 30, height climbed = 1.73 cm at ~48 h post molt), sclerotized group 1 (S1) bed bugs (n = 30, S1 = 2.42 cm at >72 h), and sclerotized group 2 (S2) bed bugs (n = 30, height climbed = 2.64 cm at >72 h post molt). When heights from all climbing events were summed, teneral bed bugs (650.8 cm climbed) differed significantly (P< 0.01) from recently sclerotized (82 cm climbed) and sclerotized (group 1 = 104.6 cm climbed, group 2 = 107.8 cm climbed) bed bugs. These findings suggested that the external surface of teneral bed bug exoskeletons possess an adhesive property. Using atomic force microscopy (AFM), we found that adhesion force of an exoskeletal (presumably molting) fluid decreased almost five-fold from 88 to 17 nN within an hour of molting. Our findings may have implications for laboratory safety and the effectiveness of bed bug traps, barriers, and biomimetic-based adhesives.
... Several studies have analyzed the effects of the blood meal source and the feeding process on mortality and fecundity (de Souza Garcia et al. 1975, Lima Gomes et al. 1990, Braga et al. 1998, Guarneri et al. 2000, Nattero et al. 2012, as well as on mean development time (Cabello et al. 1987, Martínez-Ibarra et al. 2003) of various triatomine species, and particularly for T. infestans, those effects were analyzed by Guarneri et al. (2000) and Nattero et al. (2011). Since mammal blood has a lower DNA content than bird blood, we would expect a decreasing metabolic cost and an increasing energetic efficiency in triatomines when fed on the former than on the latter (Lehane 2005). ...
Triatoma infestans (Klug, 1834) (Hemiptera: Reduviidae), the main vector of Chagas disease in South America, feeds primarily on humans, but ethical reasons preclude carrying out demographical studies using people. Thus, most laboratory studies of T. infestans are conducted using bird or mammal live hosts that may result in different demographic parameters from those obtained on human blood. Therefore, it is of interest to determine whether the use of an artificial feeder with human blood would be operational to rear triatomines and estimate population growth rates. We estimated life history traits and demographic parameters using an artificial feeder with human blood and compared them with those obtained on live hens. Both groups of T. infestans were kept under constant conditions [28 ± 1°C, 40 ± 5% relative humidity, a photoperiod of 12:12 (L:D) h] and fed weekly. On the basis of age-specific survival and age-specific fecundity, we calculated the intrinsic rate of natural increase (r), the finite rate of population growth (λ), the net reproductive rate (Ro), and the mean generation time (Tg). Our results show differences in life history traits between blood sources, resulting in smaller population growth rates on human blood than on live hens. Although demographic growth rate was smaller on human blood than on hens, it still remains positive, so the benefit/cost ratio of this feeding method seems relatively attractive. We discuss possibility of using the artificial feeder with human blood for both ecological and behavioral studies.
... Bed bugs were not selected by sex so that an accurate representation of the population could be obtained. All vials were fitted with mesh tops secured with rubber bands to allow bed bugs to feed through the mesh on an artificial feeding system (developed by Garcia et al. [27] and modified by Montes et al. [28]). All bed bugs were allowed to feed to repletion on defribinated rabbit blood (Hemostat Laboratories, Dixon, CA) by exposing vials containing bed bugs to the artificial feeding system for~30 minutes. ...
The ability of bed bugs to climb smooth surfaces is poorly documented. I observed that field strain adult bed bugs were often able to scale clean glass and metal pans. Recently molted adult bed bugs (teneral) appeared to be even more capable climbers. Climbing abilities of sclerotized adult bed bugs, teneral adult bed bugs, and recently sclerotized adult bed bugs were assessed by recording climbing heights (minimum, average, maximum) of bugs within a glass vial. Maximum climbing heights of teneral bed bugs were significantly different (p<.0083) compared to climbing heights of sclerotized bed bugs, while differences in average climbing heights approached significance (p=.09). Teneral bed bugs exhibited climbing abilities similar to all other sclerotized bed bugs after 48 hours, suggesting that climbing ability decreases as sclerotization occurs. Atomic force microscopy was conducted to examine whether this ability may be linked to molting fluid remaining on the exoskeleton, which, when combined with a more flexible cuticle, may lend an adhesive property to the surface of the exoskeleton. Preliminary AFM results indicate that the exoskeleton of teneral bed bugs is “stickier” than sclerotized bed bugs. These findings for both teneral and sclerotized bed bugs may have implications for laboratory safety and the design of interception devices.
... The experimental insects were taken from a colony of Rhodnius prolixus maintained at 28°C and 7040% relative humidity. The insects were normal mated females fed with rabbit blood at 3-week intervals, using a special feeder (Garcia et al., 1975). ...
... Adult females were fed on rabbit blood enriched with 32 Pi (10 9 cpm/ml of blood) as described elsewhere (Masuda and Oliveira, 1985) using a special feeder (Garcia et al., 1975). Five days after the blood meal, hemolymph was collected and 32 P-Lp was purified as just described for the unlabeled Lp. ...
Lipophorin is a major lipoprotein that transports lipids in insects. In Rhodnius prolixus, it transports lipids from midgut and fat body to the oocytes. Analysis by thin-layer chromatography and densitometry identified the major lipid classes present in the lipoprotein as diacylglycerol, hydrocarbons, cholesterol, and phospholipids (PLs), mainly phosphatidylethanolamine and phosphatidylcholine. The effect of preincubation at elevated temperatures on lipophorin capacity to deliver or receive lipids was studied. Transfer of PLs to the ovaries was only inhibited after preincubation of lipophorin at temperatures higher than 55°C. When it was pretreated at 75°C, maximal inhibition of phospholipid transfer was observed after 3-min heating and no difference was observed after longer times, up to 60 min. The same activity was also obtained when lipophorin was heated for 20 min at 75°C at protein concentrations from 0.2 to 10 mg/ml. After preincubation at 55°C, the same rate of lipophorin loading with PLs at the fat body was still present, and 30% of the activity was observed at 75°C. The effect of temperature on lipophorin was also analyzed by turbidity and intrinsic fluorescence determinations. Turbidity of a lipophorin solution started to increase after preincubations at temperatures higher than 65°C. Emission fluorescence spectra were obtained for lipophorin, and the spectral area decreased after preincubations at 85°C or above. These data indicated no difference in the spectral center of mass at any tested temperature. Altogether, these results demonstrate that lipophorin from R. prolixus is very resistant to high temperatures.
... Inactivated plasma and washed cells were mixed and T. cruzi cells were added (10 5 cells/ml of blood). Insects were infected by feeding in an artificial feeder as described elsewhere (Garcia et al., 1975). Some insects were dissected on the third day after feeding to check infection. ...
Quantitative real-time PCR (qPCR) has become one of the most used techniques to measure gene expression. However, normalization of gene expression data against reference genes is essential, although these are usually used without any kind of validation. The expression of seven genes was compared in organs of Rhodnius prolixus under diverse conditions, using published software to test gene expression stability. Rp18S and elongation factor 1 (RpEF -1) were the most reliable genes for normalization in qPCR when gene expression in different organs was compared. Moreover, both genes were found to be the best references when transcript levels were compared in the posterior midgut of insects infected with Trypanosoma cruzi. Rp18S was also the best reference gene in the fat bodies of unfed and fed insects. By contrast, RpEF-1 was found to be the best reference gene for comparison between posterior midguts, and RpMIP or RpActin should be used to compare gene expression in the ovaries. Although Rp18S is indicated here as the best reference in most cases, reports from the literature show that it is difficult to find an optimum reference gene. Nevertheless, validation of candidate genes to be taken as references is important when new experimental conditions are tested to avoid incorrect data interpretation.
... Bloodsucking bugs are known to probe any warm surface provided its temperature is between 32 and 45°C (Lazzari, 1990). In Rhodnius prolixus biting can be evoked only by surface temperatures between 36 and 38°C (Garcia et al., 1975). Consequently heat is sufficient to cause short range orientation in bloodsucking bugs (Nicolle and Mathis, 1941;Friend and Smith, 1971). ...
The ability of the haematophagous bug Rhodnius prolixus to approach a pure IR source only by its long-wave infrared radiation (IR) was investigated. To exclude any heated air from reaching the bug a cooled IR-transmitting window was placed between the IR-source (a Peltierelement heated to 35 degrees C) and the bug. Starved bugs were tested under invisible short-wave IR illumination (lambda<1 µm) in order to exclude also any visual cues. The number of bugs approaching the IR-source was significantly increased compared to the controls in which the IR-source was turned off (chi(2)-test, P<0.01). Our results show that Rhodnius prolixus can use infrared stimuli to find a host.
... The Rhodnius prolixus colony was maintained at 28°C and 70% r.h. at the Departamento de Bioquímica Médica (Universidade Federal do Rio de Janeiro). Normal mated females were fed on rabbit blood at 3-week intervals, using an artificial feeding apparatus (Garcia et al., 1975). ...
We have previously shown that the pathway of porphyrin synthesis operates in the blood feeding triatomine bug Rhodnius prolixus but not in the cattle tick Boophilus microplus. In the present paper we studied the correlation between heme synthesis and egg development in Rhodnius. There is a sharp increase heme biosynthetic capability in the fat body (160%) and in the ovaries (360%) in response to a blood meal, as evaluated from the activity of the enzyme delta-aminolevulinate dehydratase (EC 4.2.1.24). The in vivo inhibition of ALA-D by succinyl acetone results in a dose dependent decrease of oviposition. Oviposition is recovered when porphobilinogen, the product of the impaired reaction, is added to the succinyl acetone enriched blood. Taken together, these results show that heme biosynthesis is a fundamental event to vitellogenic females. The demand for heme in this metabolic juncture cannot be supplied by the heme eventually absorbed during blood digestion and associated with Rhodnius heme binding protein (RHBP), which is then incorporated into growing oocytes. Inhibition of heme biosynthesis results in lower levels of RHBP in the hemolymph, suggesting that the synthesis of this protein is controlled by heme availability.
The interaction between Rhodnius prolixus and Trypanosoma cruzi has huge medical importance because it responds to the transmission of Chagas disease, a neglected tropical disease that affects about eight million people worldwide. It is known that trypanosomatid pathogens depend on active lipid endocytosis from the insect host to meet growth and differentiation requirements. However, until now, knowledge on how the parasite affects the lipid physiology of individual insect organs was largely unknown. Herein, the biochemical and molecular dynamics of the triatomine R. prolixus lipid metabolism in response to T. cruzi acute infection were investigated. A qRT-PCR approach was used to determine the expression profile of 12 protein-coding genes involved in R. prolixus lipid physiology. In addition, microscopic and biochemical assays revealed the lipid droplet profile and the levels of the different identified lipid classes. Finally, spectrometry analyses were used to determine fatty acid and sterol composition and their modulation towards the infection. T. cruzi infection downregulated the transcript levels of protein-coding genes for lipid biosynthetic and degrading pathways in individual triatomine organs. On the other hand, upregulation of lipid receptor transcripts indicates an attempt to capture more lipids from hemolymphatic lipoproteins. Consequently, several lipid classes (such as monoacylglycerol, diacylglycerol, triacylglycerol, cholesteryl ester, phosphatidylcholine, and phosphatidylethanolamine) were involved in the response to the parasite challenge, although modulating only the insect fat body. T. cruzi never leaves the insect gut and yet it modulates non-infected tissues, suggesting that the association between the parasite and the vector organs is reached by cell signaling molecules. This hypothesis raises several intriguing issues to inspire future studies in the parasite-vector interaction field.
Blood-sucking insects are responsible for the transmission of several important disease-causing organisms such as viruses, bacteria, and protozoans. The hematophagous hemipteran Rhodnius prolixus is one of the most important vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. Due to the medical importance of this insect, it has been used as a study model in physiology and biochemistry since the 1930s. Artificial feeding has been recognized as a feasible and a more ethical alternative method of feeding these hematophagous insects. To prevent clotting after blood collection defibrination or treatment with anticoagulants are necessary. Although anticoagulants have been routinely used for stabilizing the collected blood, there is a gap in demonstration of the effects of using anticoagulants on the feeding and development of the hematophagous insect Rhodnius prolixus. In this study, we compared the survival rate, molting efficiency, fertility, and infection development between insects that were fed on blood containing three different anticoagulants (citrate, EDTA, and heparin). We observed that fifth instar nymphs that were fed on blood containing EDTA and citrate could not perform digestion properly, which resulted in molting inefficiency. Adult insects that were fed on EDTA-containing blood laid lower number of eggs, and also had a diminished egg hatch percentage. When we delivered T. cruzi parasites in blood containing citrate or EDTA to the insects, a lower number of parasites and metacyclic trypomastigotes was observed in the intestine compared to the group fed on heparin-containing blood. Since heparin could potentially inhibit DNA polymerase activity in DNA samples extracted from the intestine, we analyzed different heparin concentrations to determine which one is the best for use as an anticoagulant. Concentrations ranging between 2.5 and 5 U/mL were able to inhibit coagulation without severely impairing DNA polymerase activity, thus indicating that this should be considered as the range of use for feeding experiments. Our results suggest that among the three anticoagulants tested, heparin can be recommended as the anticoagulant of choice for R. prolixus feeding experiments.
Triatominae is a subfamily of the order Hemiptera whose species are able to feed in the vertebrate blood (i.e., hematophagy). This feeding behavior presents a great physiological challenge to insects, especially in Hemipteran species with a digestion performed by lysosomal-like cathepsins instead of the more common trypsin-like enzymes. With the aim of having a deeper understanding of protease involvement in the evolutionary adaptation for hematophagy in Hemipterans, we screened peptidases in the Rhodnius prolixus genome and characterized them using common blast (NCBI) and conserved domain analyses (HMMER/blast manager software, FAT, plus PFAM database). We compared the results with available sequences from other hemipteran species and with 18 arthropod genomes present in the MEROPS database. Rhodnius prolixus contains at least 433 protease coding genes, belonging to 71 protease families. Seven peptidase families in R. prolixus presented higher gene numbers when compared to other arthropod genomes. Further analysis indicated that a gene expansion of the protease family A1 (Eukaryotic aspartyl protease, PF00026) might have played a major role in the adaptation to hematophagy since most of these peptidase genes seem to be recently acquired, are expressed in the gut and present putative secretory pathway signal peptides. Besides that, most R. prolixus A1 peptidases showed high frequencies of basic residues at the protein surface, a typical structural signature of Cathepsin D-like proteins. Other peptidase families expanded in R. prolixus (i.e., C2 and M17) also presented significant differences between hematophagous (higher number of peptidases) and non-hematophagous species. This study also provides evidence for gene acquisition from microorganisms in some peptidase families in R. prolixus: (1) family M74 (murein endopeptidase), (2) family S29 (Hepatitis C virus NS3 protease), and (3) family S24 (repressor LexA). This study revealed new targets for studying the adaptation of these insects for digestion of blood meals and their competence as vectors of Chagas disease.
The Hemiptera or ′true bugs′ are widespread and numerous throughout tropical and temperate regions. Over 80 000 species are described, making this the largest of the exopterygote orders. The order has been traditionally divided into two suborders, Homoptera and Heteroptera, but a division into three or four suborders is now generally accepted. These names relate to the typical form of wings: the adults usually have the anterior pair of wings of harder consistency than the posterior pair (Hemiptera), either uniformly so (Homoptera), or with the apical portion more membranous than the remainder (Heteroptera). Many Heteroptera close their wings flat over the abdomen with the apical membranous areas overlapping (and so are sometimes referred to as ′cross-winged bugs′) (Fig. 14.2), while the Homoptera often carry their wings raised like a roof over their backs. The Homoptera are all phytophagous, and include many groups of economically important crop pests such as aphids (greenfly or plant lice), psyllids (jumping plant lice), coccids (scale insects), cicadellids (leafhoppers), and aleurodids (whitefly). Many of these are vectors of plant viruses and mycoplasmas. Sometimes such pests occur in vast numbers and, when fragmented, may become inhalant allergens.
The effect of salivarectomy on feeding in Rhodnius prolixus was studied. Salivarectomized insects drew less blood and at a lower rate than controls when fed on a rabbit. Operated insects also pierced host skin much more often (10 times in 5 min) than controls (1 or 2 times in 5 min). None of these differences was observed when feeding was performed artificially through latex membranes. Intradermal injection of salivary secretion in rat tails increased the duration of bleeding induced by small cuts made on the injection site. It is suggested that the antihaemostatic action of saliva is important for positioning of the maxillae inside blood vessels.
The effects of azadirachtin on the development of 4th-instar nymphs of Rhodnius prolixus were studied. Given through a blood meal, a dose-response relationship of azadirachtin was established using antifeedant effect and ecdysis inhibition as effective parameters. The effective dose (ED50) was 25.0 μg/ml and 4 × 10−4 μg/ml of blood, respectively, for antifeedant and ecdysis inhibition effects. Feeding inhibition is an indirect effect due to an interference of azadirachtin with the endocrine system rather than through the inhibition of chemoreceptors. Ecdysone given orally (5.0 μg/ml) and juvenile hormone analogue (70 μg/insect) counteracted the ecdysis inhibition as induced by azadirachtin.
In order to study the sensitivity of the xenodiagnosis technique a comparison between natural and artificial xenodiagnosis methods was performed in 57 chronic phase chagasic patients (31 female), with ages ranging from 26 to 83 years. All patients had demonsti-able antibodies against Trypanosoma cruzi. Forty first instar nymphs of Dipetalogaster maximus were used for each of both methods and for each patient. The positivity of xenodiagnosis artificial was significantly higher than the routine test method. These results did show that a single application of 40 bugs by the artificial method yielded a similar result than 3 applications of 40 bugs each, by the natural method.The positivity of xenodiagnosis was significantly higher in patients between 56-65 and 66-83 years old than at other ages. Males were predominant in this age group. These results showed the viability of artificial xenodiagnosis and its use in routine laboratory testing.
Lipophorin (Lp) is the main haemolymphatic lipoprotein in insects and transports lipids between different organs. In adult females, lipophorin delivers lipids to growing oocytes. In this study, the interaction of this lipoprotein with the ovaries of Rhodnius prolixus was characterised using an oocyte membrane preparation and purified radiolabelled Lp (125I-Lp). Lp-specific binding to the oocyte membrane reached equilibrium after 40-60 min and when 125I-Lp was incubated with increasing amounts of membrane protein, corresponding increases in Lp binding were observed. The specific binding of Lp to the membrane preparation was a saturable process, with a Kdof 7.1 ± 0.9 x 10-8M and a maximal binding capacity of 430 ± 40 ng 125I-Lp/µg of membrane protein. The binding was calcium independent and pH sensitive, reaching its maximum at pH 5.2-5.7. Suramin inhibited the binding interaction between Lp and the oocyte membranes, which was completely abolished at 0.5 mM suramin. The oocyte membrane preparation from R. prolixus also showed binding to Lp from Manduca sexta. When Lp was fluorescently labelled and injected into vitellogenic females, the level of Lp-oocyte binding was much higher in females that were fed whole blood than in those fed blood plasma.
The lipophorin of Rhodnius prolixus was metabolically labelled with 32P exclusively in the phospholipid moiety and purified on a potassium bromide gradient. After injection into a vitellogenic female the radioactivity from [32P]lipophorin was readily removed from the haemolymph and accumulated in several organs, including the ovary. The rate at which ovaries incorporated radioactivity from injected [32P]lipophorin varied during the days following the meal. The ability of oöcytes to take up radioactivity also varied, increasing with their sizes up to the time of chorion formation. The incorporation of radioactivity from [32P]lipophorin was several times higher than the sequestration of [14C]inulin, indicating that the process may be specific. The kinetics of incorporation were linear and the process was impaired at low temperature. Lipophorin delivered phospholipids to the growing oöcytes but its apoproteins were not accumulated.
The allatotoxic effect of 3-ethoxy-4-methoxy-6-iso-pentenylphenol on nymphal molting and metamorphosis of Rhodnius prolixus was examined. Continuous contact treatment with IPP induced the formation of precocious adults and retarded molting or initiated a permanent ecdysial stasis. Insects treated with 7-ethoxy-6-methoxy-2,2-dimethylchrornene were similarly affected. Ecdysone given orally counteracted the ecdysial stasis and also reduced the duration of the molting delay caused by IPP.
The lipophorin of adult females of Rhodnius prolixus was radioactively labelled with 32P exclusively in the phospholipid moiety and purified on a KBr ultracentrifugation gradient. The density of purified [32P]phospholipid labelled lipophorin on the fifth day after a blood meal was 1.1211 ± 0.0017 g/ml. By weight it contained 51.7% protein, 0.7% sugar and 47.6% lipid. The protein moiety was composed of three apoproteins of 226 ± 11, 86 ± 2 and 16 ± 1 kDa. Mannose and N-acetylglucosamine were the only sugars detected. Among the lipids, 66.3% were neutral lipids and 33.8% were phospholipids. Analysis by thin-layer chromatography showed that in the total phospholipids fraction 32P was distributed as follows: phosphatidylethanolamine (54.4%), phosphatidylcholine (44.7%), cardiolipin (2.1%), phosphatidylserine (0.7%), phosphatidylinositol (0.4%), sphingomyelin (0.3%) and phosphatidic acid (0.2%). The total phosphate content was 0.53 ± 0.03 nmol/μg of protein.
The protein, phospholipid and carbohydrate components of vitellin and vitellogenin from Rhodnius prolixus were analyzed after being labelled by feeding the insects with blood enriched with 32Pi. The carbohydrate moiety is comprised mainly of mannose residue (90%), but also contains glucose and traces of galactose and glucosamine. Both vitellin and vitellogenin are present in two different aggregational states that differ in molecular weight. Each of the individual aggregates releases, under denaturing conditions, the same four polypeptides with approximate molecular weights of 180, 158, 44 and 38 K daltons with no consistent stoichiometries. Analysis of the phosphorylated yolk protein after specific precipitation with serum against vitellin showed 62% of the total radioactivity in the phospholipid fraction and 38% in the glycoprotein fraction. Upon further analysis by thin layer chromatography, the following phospholipids were found: phosphatidylcholine (64.2%), phosphatidylethanolamine (30%), phosphatidic acid (4,4%), sphingomyelin (0.7%), phosphatidylserine (0.6%) and cardiolipin (0.2%). Analysis of the glycoprotein fraction showed that all four subunits were phosphorylated, part of the radioactivity being found as [32P]serine and [32P]mannose.
Dietary [22,23-3H2]dihydroazadirachtin A, fed in a blood meal to Rhodnius prolixus, was absorbed, transported by the haemolymph, retained, and excreted unmetabolized by the organism. In the haemolymph, the level of labelled compound rose steeply to a peak 24 h after feeding, and then declined gradually. Dihydroazadirachtin A was rapidly excreted, and the highest label elimination was found during 12–24 h after feeding. A constant quantity of dihydroazadirachtin A was recovered, from the head, visceral structures and the remaining body 5 and 10 days after ingestion. The highest amount per mg tissue was found in the Malpighian tubules and midgut, the lowest in the head and the remaining body. Pure urine contained the unchanged labelled compound demonstrating the excretion of unmetabolized dihydroazadirachtin A through the Malpighian tubules.
Antibacterial activity induced in the haemolymph of adult Rhodnius prolixus was investigated. Little or no antibacterial activity appeared in the first hours after Streptococcus mutans inoculation, but the activity increased reaching a maximum 5–6 days later and then declined gradually. The bacteria were destroyed in the haemolymph when the level of the antibacterial activity attained maximal value. The antibacterial activity appears to be related to the defence mechanism of the insect against bacterial infection. The activity was semi-purified by one-step Bio-Gel P-10 Gel-filtration and the estimated apparent mol. wt was 7,000 Daltons. It was found that this activity was due to a protein of small molecular weight. The activity was heat-stable, dialyzable and inactivated by trypsin treatment. The addition of haemolymph, obtained after inoculation of S. mutans into insects, to label-DNA growing cells (1) caused a dramatic reduction in the viable cells, measured by colony-forming units, and (2) increased approx 4-fold the releasing of labelled-DNA into the supernatant of the bacterial culture media. The possibility of this activity being lysozyme was excluded. The possible mechanism of action of this antibacterial activity is discussed.
Subcellular fractions of the cells of the posterior midgut of Rhodnius prolixus nymphs were obtained by conventional homogenization, under isotonic or hypotonic conditions, followed by differential centrifugation. Alkaline phosphatase and membrane-bound α-mannosidase are more abundant in fractions in which vesicles displaying brush-borders predominate. α-Glucosidase is associated with large membranous structures, although its subcellular distribution is different from that of alkaline phosphatase and α-mannosidase. α-Mannosidase-carrying membranes were resolved from α-glucosidase-carrying membranes in sucrose gradients, supporting the hypothesis that these enzymes are part of respectively, protein-rich inner and protein-poor outer microvillar membranes. To account for the soluble enzyme activities that sediment with vesicles displaying brush borders, major amounts of aminopeptidases are assumed to be trapped in the space between outer and inner microvillar membranes, from where they are set free by homogenization and (or) freezing and thawing. There are at least three different aminopeptidases, based on their activities toward several substrates and on sedimentation data. The results favor the view that oligomers derived from partial digestion of polymeric food are hydrolyzed down to monomers by enzymes trapped between microvillar membranes or on the surface of midgut cells. The use of microvillar membranes as a peritrophic membrane by R. prolixus is thought to be a derived character evolved from a putative phloem feeder Hemiptera ancestor.
32P-Labelled midguts (32P-midguts) of Rhodnius prolixus females were incubated in the presence of nonradioactive purified lipophorin and the release of radioactivity to the medium was analysed. The radioactivity found in the medium was associated with lipophorin phospholipids. When the 32P-midguts were incubated in the absence of lipophorin, no 32P-phospholipids were found in the medium. Comparative analysis by thin-layer chromatography of 32P-phospholipids derived from metabolically labelled 32P-midgut or lipophorin particles after incubation with 32P-midgut showed some differences, revealing a possible selectivity in the process of phospholipids transfer. The transfer of phospholipids to lipophorin was linear with time up to 45 min, was saturable with respect to the concentration of lipophorin, and was half-maximal at about 5 mg/ml. The binding of 32P-lipophorin to the midgut at O°C reached the equilibrium at about 1 h of incubation. The binding of 32P-lipophorin was inhibited by an excess of nonradioactive lipophorin, which suggests a specific receptor for lipophorin. The capacity of midguts and fat bodies to transfer phospholipids to lipophorin varied during the days following the meal. When lipophorin enzymatically depleted of phospholipids by treatment with phospholipase A2 was incubated with 32P-midguts, the same amount of phospholipids was transferred, indicating a net gain of phospholipids by the particle. © 1995 Wiley-Liss, Inc.
The growth of oocytes was severely reduced in female Rhodnius prolixus treated with azadirachtin A (AZA). When administered in a blood meal, AZA (1 μg/ml) inhibited phospholipid transfer to the ovaries without altering the availability of yolk protein in the hemolymph or its uptake by 1 mm oocytes. The lipid composition of lipophorin, its concentration in the hemolymph, and its density were normal in AZA-treated females. Partial inhibition of phospholipid transfer was observed when lipophorin from AZA-treated females was injected into normal females, or when lipophorin from normal females was injected into AZA-treated females. The two effects were additive, so that the transfer of phospholipids from the lipophorin of AZA-treated females to the oocytes of AZA-treated females was nearly eliminated. In combination, these effects of AZA on phospholipid transfer to the oocytes limit the capacity of AZA-treated females to produce eggs. © 1994 Wiley-Liss, Inc.
1.1. The properties of β-acetylglucosaminidase, acid phosphatase, aminopeptidase, cathepsin B-like proteinase, α-galactosidase, α-glucosidase, detergent-solubilized membrane-bound α-glucosidase, β-glucosidase, α-mannosidase and detergent-solubilized membrane-bound α-mannosidase from posterior midgut of Rhodnius prolixus adults were determined by density-gradient centrifugation and kinetic methods.2.2. The posterior midgut hydrolases from R. prolixus adults and nymphs were found to be very similar.3.3. The results lend support to the hypothesis that digestive enzymes are, in contrast to holometabolous insects, identical in young and adult forms of hemimetabolous insects.
The insect Rhodnius prolixus synthesizes chondroitin sulfate and heparan sulfate after a blood meal containing radioactive 35S-inorganic sulfate. A 3.5-fold enhancement of chondroitin sulfate synthesis was obtained when the meal was supplemented with 10−3 M whereas no significant changes were observed for heparan sulfate. Time course experiments have shown that the synthesis of chondroitin sulfate in the presence of phenylxyloside is maximum 2 days after feeding and decreases slowly after 10 days, whereas heparan sulfate remains constant during the whole period. The analysis of this chondroitin sulfate has shown that its structure is similar to the chondroitin sulfate synthesized by mammalian cells in culture in the presence of phenylxyloside. It is concluded that the synthesis of these compounds follows the same pattern of mammals and that their structures are maintained through evolution.
A haemolytic factor, which lysed sheep red cells in an isotonic buffer, was found in the crop of all larval stages and adult Rhodnius prolixus. Little or no haemolytic factor occurred in unfed insects but haemolytic activity increased for 2–4 days after feeding. From the 4th day on, the activity declined gradually. Fifth-instar larvae fed on whole blood, erythrocytes and haemoglobin produced large quantities of haemolytic factor, while those fed on plasma and erythrocyte stroma did not. The haemolytic factor was purified approximately 1200-fold by a two-step procedure: (1) Bio-Gel P-6 Gel-Filtration and (2) SP-Sephadex chromatography. Purified haemolytic factor was heatstable (100°C, 10 min), dialysable, inactivated by trypsin treatment, and could be recovered in the supernatant after addition of ethanol. It was concluded that the haemolytic factor is a peptide displaying a basic character.
the regulation of haemolymph titres of ecdysteroids during larval development of the bloodsucking bug, Rhodnius prolixus was studied. Corpus allatum ablation in 4th-instar larvae 1 day after feeding was reflected in an increase of the intermoult period and in a high level of ecdysial arrest. These effects could be corrected by juvenile hormone and ecdysone therapies. Comparison of the ecdysteroid titres in haemolymph determined in control and allatectomized larvae, at different intervals after feeding, showed that allatectomy drastically depressed the ecdysteroid levels. Juvenile hormone treatment reestablished ecdysteroid titres in the haemolymph of allatectomized insects. Isolated prothoracic glands from allatectomized larvae had a very low production of ecdysteroid-RIA-activity when compared with prothoracic glands from control or allatectomized larvae which received in vivo juvenile hormone treatment. The complexity of the corpus allatum-prothoracic glands interaction in Rhodnius post-embryonic development is discussed.
Groups of four Rhodnius prolixus larvae in the 5th-instar received no food or were given once only through a special feeder, similar weights of food solutions. These were either protein solutions (whole human blood, its plasma or erythrocytes, egg albumin, bovine serum albumin, haemoglobin) or non-protein solutions (saline, casein hydrolysate, dextran, maltose). Following feeding, the protein content and caseinolytic activity of the larval midgut homogenates, determined between the 3rd and 14th days, were practically the same in starved larvae and in larvae receiving non-protein solutions. In the protein-fed larvae the protein content and the specific protease activity increased. These findings exclude neurosecretory control and indicate that ingested protein stimulate the proteolytic activity of R. prolixus midgut.
A calcium dependent apyrase activity (ATP→AMP + 2Pi) has been characterized in the salivary secretion of Rhodnius prolixus. High levels of this activity were found in the crop of all stages of larvae and the adults after a single blood or saline meal. The activity persisted for several days but was totally absent in the crop insects from which the salivary glands had been removed. The use of this activity as a saliva marker shows that the insect salivates during the whole meal and most of the saliva is ingested with the food. The physiological role of this activity is discussed. A simple method for saliva collection and a technique for the surgical ablation of the salivary glands in adult insects are described.
The uptake of yolk protein in Rhodnius prolixus was studied in vivo and in vitro using a metabolically labelled [32P]yolk protein purified on a potassium bromide gradient. The [32P]vitellin is readily removed from haemolymph and specifically accumulates in the ovary. The ability of oöcytes to take up yolk protein at different stages of development increases with their size up to the time of chorion formation. This increase in uptake capacity of oöcytes correlates with the opening of intercellular space in the follicular epithelium. The rate of uptake is dependent on the external concentration of yolk protein and temperature, and can be saturated. Separate analysis of binding shows a similar dependence on yolk protein concentration. Soon after a meal, the vitellogenin concentration increases in the haemolymph until it reaches a steady-state concentration at which the rate of vitellogenin uptake matches the rate of vitellogenin synthesis. Most ovarian growth occurs while the concentration of vitellogenin in the haemolymph is at this steady-state level, which is in turn very close to its ovarian binding constant of 12 mg/ml determined in vitro. Thus it is suggested that the uptake system normally works undersaturated and below its maximal rate of uptake.
1.1. 1-14C Oleic acid and glyceryl-tri-1-14C oleate were fed to Triatoma infestans and their digestion, absorption and incorporation in hemolymph lipids, were investigated.2.2. Four hours after feeding labeled oleic acid, significant radioactivity was only found in midgut and hemolymph lipids. Little labeling was observed in the fat body at this time and significant radioactivity was only recovered after 8 hr.3.3. Four hours after feeding, 14C oleic acid was largely incorporated to the diacylglycerols of the hemolymph high density lipoprotein (HDL) () indicating that this lipoprotein is the principal carrier of absorbed fatty acids.4.4. Glyceryl-tri-1-14C oleate was hydrolyzed in midgut and the resulting fatty acids were absorbed and transported by the hemolymph as diacylglycerol molecules.
Soluble β-acetylglucosaminidase, α-galactosidase, α-glucosidase and α-mannosidase predominate in anterior midgut lumen; soluble aminopeptidase, membrane-bound α-glucosidase and membrane-bound α-mannosidase, in posterior midgut tissue; and proteinases, sb-glucosidase and β-mannosidase, in posterior midgut lumen. Ultracentrifugation data suggest that there is only one major molecular species of the following soluble enzymes: aminopeptidase (Mr 61,000), with a broad specificity toward aminoacyl-β-naphthylamides; β-glucosidase (Mr 64,000), active on several β-glycosides; and acid phosphatase (Mr 82,000), which hydrolyzes many substrates and is competitively inhibited by tartrate (Ki 2 μM), molybdate (Ki 9 nM) and fluoride (three ligands per active site). There are major (85% of total) and minor (15%) thiol-activated hemoglobin hydrolase resolved by ultracentrifugation. The major activity (Mr 40,000) is cathepsin B-like hydrolyzing , whereas the minor activity (Mr 88,000) is cathepsin d-like, being more active at pH 3.5 and being inhibited by pepstatin. The symbiont, Nocardia rhodnii, possesses an amylase which is found in Rhodnius prolixus midgut and an aminopeptidase and a BAPA hydrolase different from those of R. prolixus. The results suggest that digestion starts in the lumen of posterior midgut and ends with the action of enzymes trapped between extracellular membranes or on the surface of midgut cells. The use of cathepsin-like proteases is thought to be derived character evolved from a putative seed-sucker ancestor.
Triatomine insects are obligatory blood-feeders that detoxify most of the hemoglobin-derived heme through its crystallization into hemozoin (Hz). Previous evidence demonstrates the key role of midgut perimicrovillar membranes (PMVM) on heme crystallization in triatomines. Here, we investigated some of the physico-chemical and physiological aspects of heme crystallization induced by Rhodnius prolixus PMVM. Hz formation in vitro proceeded optimally at pH 4.8 and 28 degrees C, apparently involving three kinetically distinct mechanisms along this process. Furthermore, the insect feeding status and age affected PMVM-induced heme crystallization whereas pharmacological blockage of PMVM formation by azadirachtin, reduced hemoglobin digestion and Hz formation in vivo. Mössbauer spectrometry analyses of R. prolixus midgut showed that Hz represents the only measurable iron species found four days after a blood meal. Autocatalytic heme crystallization to Hz is revealed to be an inefficient process and this conversion is further reduced as the Hz concentration increases. Also, PMVM-derived lipids were able to induce rapid Hz formation, regardless of the diet composition. These results indicate that PMVM-driven Hz formation in R. prolixus midgut occurs at physiologically relevant physico-chemical conditions and that lipids derived from this structure play an important role in heme crystallization.
Rhodnius pictipes (Hemiptera, Reduviidae) from Serra Norte, State of Pará, Brazil, aclimatized in an insectary at the Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Departamento de Entomologia, Instituto Oswaldo Cruz, were fed through a silicone membrane. In order to know the viability and the efficiency of this membrane compared with insects fed on mice, the number of bloodmeals taken, period of development of the five nymphal instars, longevity of adults, average amount of blood intake in each meal and percent of mortality were observed. A total of 310 insects, were used, comprising 50 nymphs of each instar, as well as 30 male and 30 female adults. Insects fed artificially had reduced minimal and maximal periods of development than the group fed on mice. The largest relative increase of body weight was observed in the 2nd instar followed by the 1st, and the amount of blood ingested increased during the development, to the 5th instar for both groups. There were no significant differences between the groups fed artificially and in vivo according to Tukey's test for p>0.05. The percent of mortality in the 1st instar was 18% for artificially fed and 16% for the group fed on mice; these percentages decreased as insects developed until the 4th instar, without mortality, returning to increase in the 5th instar. R. pictipes was shown to be easily adaptable to artificial feeding, and could be considered as an important and viable experimental model.
Oviposition and oögenesis can be inhibited in female Rhodnius prolixus by ecdysone given by the digestive tract. The inhibition is dose-dependent, and doses higher than 4.0 ng ecdysone/mg body weight drastically reduce the size and shape of the whole ovaries. In ecdysone-treated insects, normal oviposition and oögenesis can be re-established by a subsequent blood meal without ecdysone, or by the application of a juvenile hormone analogue.These results suggest that ecdysone inhibits juvenile hormone production.
The lipophorin of Rhodnius prolixus metabolically labelled with 32P exclusively in the phospholipid moiety was purified on a potassium bromide gradient and treated with phospholipase A2 in the presence of an excess of fatty acid-free albumin. The treatment completely removed the phospholipids from the particles and generated [32P]-lysophosphatidylcholine, [32P]-lysophosphatidylethanolamine, and free fatty acids that remained bound to albumin. The phospholipid-depleted lipophorin particles remained soluble, indicating that phospholipids are not essential in maintaining the stability of the particles in aqueous solution. Complete removal of phospholipids did not affect the association of apolipophorin III with lipophorin particles. Lipophorin density increased slightly from 1.120 to 1.134 g/ml after treatment. The phospholipid-depleted particles also retained their ability to be recognized and loaded in vitro with phospholipids delivered by the fat body, thus supporting the concept of lipophorin's role as a reusable lipid shuttle for phospholipids.
The effects of precocene II (10 to 300 μg/ml of blood), given orally, on the development of third- and fourth-instar larvae of Rhodnius prolixus were studied. Precocene acted as a feeding deterrent, reducing the amount of blood eaten, and caused a high mortality at high doses. Molting was delayed in a dose-dependent manner and in several cases ecdysis was completely prevented. Large numbers of insects underwent precocious metamorphosis. A significant correlation between the prolongation of the molting cycle and morphogenetic adultoid characteristics was observed. Ecdysone given orally (5 μg/ml of blood) counteracted the ecdysis prevention and also reduced the duration of the molting cycle which is prolonged by precocene II. However, ecdysone did not preclude the adultiform production. Precocious adults with slight imaginal characters were able to feed and initiate the next ecdysis. Adultoids with distinct imaginal characteristics were short-lived and did not feed despite repeated opportunities.
A novel calcium-binding phosphoprotein was isolated from the oocytes of the blood-sucking bug Rhodnius prolixus. This protein exhibits an apparent molecular mass of 18 kDa on gel filtration, but migrates as an 8-kDa band on N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine/SDS-polyacrylamide gels. It has a high content of serine (24% of the total
number of residues), and phosphoserine is the sole amino acid phosphorylated in vivo. A similar protein was partially purified from the hemolymph. It resembles the oocyte form of the protein in its NH2-terminal sequence and its ability to be taken up by growing ovaries. 45Ca binding to the oocyte phosphoprotein was determined after SDS-polyacrylamide gel electrophoresis followed by blotting on
nitrocellulose membranes. Titration of Ca2+-binding sites shows a high capacity (≅50 mol/mol of protein), but a low affinity (K0.5 ≅ 10−3 M). Based on these characteristics, we have named this protein Rhodnius calcium-binding phosphoprotein. It resembles phosvitin, a phosphoprotein present in the oocytes of nonmammalian vertebrates.
The infection pattern in Swiss mice and Triatomine bugs (Rhodnius neglectus) of eleven clones and the original stock of a Trypanosoma cruzi isolate, derived from a naturally infected Didelphis marsupialis, were biochemically and biologically characterized. The clones and the original isolate were in the same zymodeme (Z1) except that two clones were found to be in zymodeme 2 when tested with G6PDH. Although infective, neither the original isolate nor the clones were highly virulent for the mice and lesions were only observed in mice infected with the original stock and one of the clones (F8). All clones and the original isolate infected bugs well while only the original isolate and clones E2 and F3 yielded high metacyclogenesis rates. An observed correlation between absence of lesions in the mammal host and high metacyclogenesis rates in the invertebrate host suggest a evolutionary trade off i.e. a fitness increase in one trait which is accompanied by a fitness reduction in a different one. Our results suggest that in a species as heterogeneous as T. cruzi, a cooperation effect among the subpopulations should be considered.
Malaria parasites digest haemoglobin and detoxify the free haem by its sequestration into an insoluble dark-brown pigment known as haemozoin (Hz). Until recently, this pigment could be found only in Plasmodium parasites. However, we have shown that Hz is also present in the midgut of the blood-sucking insect Rhodnius prolixus [Oliveira et al. (1999) Nature 400, 517-518]. Here we show that Hz synthesis in the midgut of this insect is promoted by a particulate fraction from intestine lumen. Haem aggregation activity is heat-labile and is inhibited in vitro by chloroquine (CLQ). Inhibition of Hz formation in vivo by feeding insects with CLQ leads to increased levels of haem in the haemolymph of the insect, which resulted in increased lipid peroxidation. Taken together, these results indicate that a factor capable of promoting Hz crystallisation is present in R. prolixus midgut and that this activity represents an important physiological defence of this insect against haem toxicity.
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